Method and apparatus for high performance wide field photothermal imaging and spectroscopy

ABSTRACT

A system for infrared analysis over a wide field area of a sample is disclosed herein that relies on interference of non-diffractively separated beams of light containing image data corresponding to the sample, as well as a photothermal effect on the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional PatentApplication 62/968,900, filed Jan. 31, 2020, the contents of which areincorporated in full herein by reference.

TECHNICAL FIELD

Embodiments disclosed herein relate to investigating or analyzingmaterials by the use of optical systems, (i.e., using infrared, visible,or ultraviolet light). Embodiments described herein relate to imagingand spectroscopy, and, more particularly, to enhancements tophotothermal imaging and spectroscopy systems and techniques foracquiring spectral information indicative of the optical propertiesand/or material or chemical composition of a sample, for example,information that correlates to an infrared (IR) absorption spectrum.

BACKGROUND

Fourier Transform Infrared (FTIR) spectroscopy is the most common formof IR spectroscopy. FTIR works by measuring transmission of an infraredlight through a sample or reflection of IR light from a sample as afunction of wavenumber (a measure of the frequency of the IR light).FTIR based microscopes combine an FTIR spectrometer and microscopeoptics to provide spatially resolved measurements of IR absorption,transmission, and/or reflection. A bedrock physical constraint onconventional FTIR microscopy is that it can only achieve spatialresolution on the order of the wavelength of the IR light used. Thefundamental limit is determined by optical diffraction and is set by thewavelength of the IR light and the numerical aperture of the IRillumination and/or collection optics. Practical limitations may degradethis spatial resolution further. The spatial resolution of the FTIRmicroscope is wavelength dependent, but is on the order of 10 micronsfor wavelengths in the mid-IR region (corresponding to wavelengthsgreater than about 2 microns). An example of an FTIR spectroscopyapproach is shown, for example, in U.S. Pat. No. 7,630,081, whichdescribes recent improvements to FTIR interferometers. Conventional FTIRspectroscopy can involve significant sample preparation to ensureappropriate transmission of the mid-IR beam through the sample, which isnot practicable or desirable for many opaque, frangible, or biologicalsubstances.

Attenuated Total Reflection (ATR) spectroscopy is based on reflection ofa beam through an intervening crystal in direct contact with the sample.ATR spectroscopy can achieve somewhat higher spatial resolution thantransmission FTIR but requires direct contact of the intervening crystalwith the sample which can cause deformation, breaking of the sample, andmeasurement variability due to the quality of the contact. Both FTIR andATR suffer from a variety of artifacts that can distort the spectra,including size and shape dependent scattering artifacts and dispersiveeffects, especially when operated in reflection. These issues can makeit very difficult to compare spectra to FTIR library spectra, thuscomplicating material identification and/or quantification.

Raman spectroscopy is based on illuminating a sample with a narrow bandlaser source and measuring the spectrum of wavelength shifted light thatscatters from the illuminated area. Raman spectroscopy can achieveresolutions as low as a few hundred nanometers in theory, but usuallyhas a practical limit of several hundred nanometers or more. An earlyexample of a Raman spectroscopy approach is shown, for example, in U.S.Pat. No. 2,940,355. Although Raman spectroscopy can achieve resolutionsin the hundreds of nanometers range, it also has limitations based onvariability of sample fluorescence and much smaller spectral librariesthan are available using FTIR.

U.S. Pat. No. 9,091,594 describes an alternative non-destructiveapproach for photothermal spectroscopy for chemical spectroscopy andimaging that uses two beams of light of differing wavelengths to achievesub-micron spatial resolution, but in a non-contact manner and withoutthe onerous sample preparation requirements associated with FTIRtechniques described above. One method described in that patent includesilluminating a sample with a first beam of IR light having a wavelengthof at least 2.5 microns to create a photothermal change in a regionwithin the sample due to absorption of energy from the first beam, andthen illuminating at least a portion of the region within the samplewith a second beam of light having a wavelength of less than 2.5 micronsto detect the photothermal change in the region at a resolution smallerthan a diffraction limit of the first beam.

Quantitative Phase Imaging (QPI) is a technique that seeks to extractquantitative measurements of optical phase for optical microscopyapplications. Useful review articles on the subject include: (1) BasantaBhaduri, Chris Edwards, Hoa Pham, Renjie Zhou, Tan H. Nguyen, Lynford L.Goddard, and Gabriel Popescu, “Diffraction phase microscopy: principlesand applications in materials and life sciences,” Adv. Opt. Photon. 6,57-119 (2014), https://doi.org/10.1364/AOP.6.000057; and (2) Park, Y.,Depeursinge, C. & Popescu, G. Quantitative phase imaging in biomedicine.Nature Photon 12, 578-589 (2018) doi:10.1038/s41566-018-0253-x, both ofwhich are hereby incorporated by reference.

One form of QPI has been combined with infrared spectroscopy asdescribed in Miu Tamamitsu, Keiichiro Toda, Ryoichi Horisaki, and TakuroIdeguchi, “Quantitative phase imaging with molecular vibrationalsensitivity,” Opt. Lett. 44, 3729-3732 (2019),https://doi.org/10.1364/OL.44.003729, hereby incorporated by reference.While this combination does permit wide field infrared spectroscopyusing a QPI based approach, the use of diffractive optics to createinterfering sample and reference beams results in a large portion of thelight containing sample information to be discarded, thus constrainingcamera frame rates, reducing signal-to-noise ratio, and/or requiringlengthy data collection times.

Phase contrast microscopy is a well-established technique in opticalmicroscopy (see for example M. Pluta, Advanced light microscopy. Vol. 1,chapter 5, Amsterdam: Elsevier, 1988). Phase contrast microscopy isgenerally used for creating amplitude (brightness) contrast on highlytransparent samples, (e.g., biological cells that produce minimalcontrast in brightfield microscopy). Even though biological cells absorbvery little light, resulting in minimal brightness contrast, they doincur a significant optical phase change. Phase contrast microscopy isoften used to convert the phase shifts induced by biological and othermaterials into brightness contrast that can then be seen by eye or by acamera. Traditional phase contrast microscopy provides challenges forquantitative analysis of optical phase differences because of variousartifacts including complex nonlinear dependence of brightness on sampleheight, contrast inversions, halo artifacts and other issues. On theother hand, phase contrast microscopy is extremely widely used andavailable on many thousands of research microscopes around the world.Providing a technique to perform infrared spectroscopy on such a widelydistributed platform therefore offers significant benefits. Infraredspectroscopy has also been combined with conventional phase contrastoptical microscopy as described in Toda, K., Tamamitsu, M., Nagashima,Y. et al. Molecular contrast on phase-contrast microscope. Sci Rep 9,9957 (2019) doi:10.1038/s41598-019-46383-6, hereby incorporated byreference. The challenges associated with quantifying measurements inconventional phase contrast microscopy, however, also complicateinterpretation of IR absorption signals inferred by conventional phasecontrast microscopy. Specifically, nonlinear dependence on sample height(thickness), contrast inversion, halo artifacts and other issues canaffect the sensitivity of the measurement of IR absorption and can causedistortions in IR spectra and chemical images obtained by thistechnique. For example the supplementary information in the article byToda cited above in this paragraph describes the presence of a “spuriousnegative signal” that creates distortions in photothermal images whenusing conventional phase contrast microscopy.

Methods and apparatuses described herein provide improved performanceand overcome many of the limitations of prior instruments for infraredspectroscopic analysis.

SUMMARY

Systems and methods are disclosed herein for infrared analysis over awide field area of a sample. In an embodiment, a system includes aninfrared source configured to illuminate a region of the sample with apump beam of infrared radiation to create in infrared illuminatedregion; a probe radiation source configured to generate a probe beamthat illuminates a wide field region of the sample wherein the widefield region is at least 50 microns in diameter and at least partiallyoverlaps the infrared illuminated region of the sample; a focusing opticarranged to collect the probe beam from the sample; a first opticalsystem comprising a non-diffractive beam splitter that divides the probebeam collected from the sample onto at least two paths, a first path fora reference beam and a second path for a sample beam; a second opticalsystem comprising a 4f optical relay system and arranged to spatiallyfilter the reference beam and create an inteferogram formed between thereference beam and the sample beam as part of an image of the region ofthe sample on a surface of an array detector that is captured as animage frame of the wide field region of the sample; and an analyzerconfigured to analyze the image frame to determine signals indicative ofphotothermal infrared absorption over the wide field area of the sample.

In another embodiment, a system for infrared analysis over a wide fieldarea of a sample includes an infrared source configured to illuminate aregion of the sample with a pump beam of infrared radiation to create ininfrared illuminated region; a probe radiation source configured togenerate a probe beam that illuminates a wide field region of the samplewherein the wide field region is at least 50 microns in diameter and atleast partially overlaps the infrared illuminated region of the sample;a focusing optic arranged to collect the probe beam from the sample; afirst optical system comprising a non-diffractive beam splitter thatdivides the probe beam collected from the sample onto at least twopaths, a first path for a reference beam and a second path for a samplebeam; a second optical system comprising a 4f optical relay system andarranged to spatially filter the reference beam and create aninterferogram formed between the reference beam and the sample beam aspart of an image of the region of the sample on a surface of an arraydetector that is captured as an image frame of the wide field region ofthe sample; and an analyzer configured to analyze the image frame todetermine signals indicative of photothermal infrared absorption overthe wide field area of the sample, wherein the array detector is acamera and the first optic system and the second optic system areconfigured to provide an optical throughput efficiency of at least 50%.

In a third embodiment, a system for infrared analysis over a wide fieldarea of a sample includes an infrared source configured to illuminate aregion of the sample with a pump beam of infrared radiation to create ininfrared illuminated region; a probe radiation source configured togenerate an annular probe beam that illuminates a wide field region ofthe sample wherein the wide field region is at least 50 microns indiameter and at least partially overlaps the infrared illuminated regionof the sample; a focusing optic arranged to collect the probe beam fromthe sample; an optical system comprising a 4f optical relay systemincluding at least one variable phase retarder configured with anannular phase shift pattern to create phase contrast interferencebetween direct/surround illumination probe light that passes through thesample with probe light scattered by the sample to create aninterference image on a surface of an array detector that is captured asan image frame of the wide field region of the sample; and an analyzerconfigured to analyze the image frame to determine signals indicative ofphotothermal infrared absorption over the wide field area of the sample.

The details of one or more aspects of the disclosure are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the techniques described in this disclosurewill be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and advantages of the embodiments provided herein are describedwith reference to the following detailed description in conjunction withthe accompanying drawings. Throughout the drawings, reference numbersmay be re-used to indicate correspondence between referenced elements.The drawings are provided to illustrate example embodiments describedherein and are not intended to limit the scope of the disclosure.

FIG. 1 is a schematic diagram of a system for a high-performance widefield photothermal IR spectroscopy and imaging using a non-diffractivebeam splitter, according to an embodiment.

FIGS. 2A and 2B depict light paths of illumination light and imaginglight, respectively, in an embodiment of a high-performance wide fieldphotothermal IR spectroscopy and imaging system.

FIGS. 3A-3C are depictions of phase-separated signal analysis techniquesfor wide field photothermal IR spectroscopy and imaging, according to anembodiment.

FIG. 4 illustrates the results of a calculation performed according tothe techniques described in FIG. 3 .

FIG. 5 is a schematic diagram of a high-performance wide fieldphotothermal IR spectroscopy and imaging system employing a non-commonpath interferometer, according to an embodiment.

FIG. 6 is a schematic diagram of a high-performance wide fieldphotothermal IR spectroscopy and imaging system employing a non-commonpath quadrature interferometer, according to an embodiment.

FIG. 7 is a schematic diagram of another system for a high-performancewide field photothermal IR spectroscopy and imaging using anon-diffractive beam splitter, according to an embodiment.

FIG. 8 is a schematic diagram of another system for a high-performancewide field photothermal IR spectroscopy and imaging using phase contrastmicroscopy with a spatial light modulator to overcome sensitivityvariations, according to an embodiment.

FIG. 9 is a schematic diagram of another system for a high-performancewide field photothermal IR spectroscopy and imaging using anon-diffractive beam splitter in a reflection mode configuration,according to an embodiment.

FIG. 10 is a schematic diagram of another system for a high-performancewide field photothermal IR spectroscopy and imaging using phase contrastmicroscopy with a variable phase interferometer, according to anembodiment.

FIG. 11 is a schematic diagram of a timing diagram related to theembodiment of FIG. 10 .

While various embodiments are amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in detail. It should beunderstood, however, that the intention is not to limit the claimedinventions to the particular embodiments described. On the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the subject matter as defined bythe claims.

DETAILED DESCRIPTION Definitions

For purposes of this specification, the following terms are specificallydefined as follows:

An “analyzer/controller” refers to a system to facilitate dataacquisition and control of the photothermal IR spectroscopy system. Theanalyzer/controller may be a single integrated electronic enclosure ormay comprise multiple distributed elements. The control elements mayprovide control for positioning and/or scanning of the fiber probeand/or sample. They may also collect data about the probe beamintensity, motion, optical phase or other response, provide control overthe excitation and/or probe power, polarization, steering, focus and/orother functions. The control elements etc. may include a computerprogram method or a digital logic method and may be implemented usingany combination of a variety of computing devices (computers, PersonalElectronic Devices), analog and/or digital discrete circuit components(transistors, resistors, capacitors, inductors, diodes, etc.),programmable logic, microprocessors, microcontrollers, single boardcomputers, application-specific integrated circuits, or other circuitelements. A memory configured to store computer programs may beimplemented along with discrete circuit components to carry out one ormore of the processes described herein.

“Beam splitter” refers to an optical element that can divide light ontoat least two paths. A beam splitter can comprise a plate, a cube and/ora prism or other shapes/configurations that can divide a beam of light.A “non-diffractive beam splitter” is a beam splitter that does not use adiffraction grating or a diffraction pattern to divide the beams. Thebeam splitter can comprise a thin film that is partially reflecting atthe wavelength of interest such that a portion of an incident beam isreflected and another portion is transmitted. A beam splitter may bepolarizing, wherein in substantially transmits light of one polarizationand reflects light of an orthogonal polarization. A beam splitter mayalso divide light along two transmission paths based on polarization,for example in the case that the beam splitter is a Rochon, Nomarski orWollaston prism that divide light onto paths separated by a small angleon the basis of polarization. Another example is a polarizing beamsplitter cube which divides light of orthogonal polarization onto twopaths separated by 90 degrees. A beam splitter may also benon-polarizing, where light is divided between two paths withoutsubstantial dependence on the polarization of the incident light. A beamsplitter can also be an optical fiber-based device, for examplesplitting light from one input optical fiber into at least two outputoptical fibers, for example a 1×2 fiber coupler. A beam splitter may bea 50:50 beam splitter in which substantially equal fractions of lightare directed on two different paths. They can also be unbalanced, forexample a 90:10 or 70:30 or similar beam splitter that direction 90% oflight on one path and 10% on another, or 70% on one path and 30% onanother. Note that a beam splitter can also be used to combine two beamsonto the same optical path, i.e. combining one beam that reflects off ofthe beam splitter interface with another beam that is transmittedthrough the beam splitter interface. A beam splitter cube, for example,can be used as both a beam splitter and a beam combiner. For example, aMach-Zehnder interferometer uses one beam splitter to split incidentlight onto two paths and a second beam splitter to recombine the twobeams. In this case, the second beam splitter is being used as a beamcombiner. In a Michelson interferometer, a single beam splitter is usedto both divide the incident light and then recombine it. Thus, the beamsplitter in a Michelson interferometer as being used as both a beamsplitter and a beam combiner. A beam splitter/combiner can also be anoptical fiber-based device, for example splitter or combining the lightfrom two input fibers into one output fiber, for example a 1×2 fibercoupler. A single 1×2 fiber coupler can be used as both a beam splitterand a beam combiner.

A “camera” refers to an array-based photodetector comprising a pluralityof photosensitive pixels. A camera may comprise one or more technologyincluding but not limited to CCD, EM-CCD, CMOS, s-CMOS, and/or otherphotosensitive array technologies. The camera may support frame ratesfrom a few frames per seconds, hundreds of frames per second, or eventhousands of frames per second or higher. “Collecting probe light,”“Collecting probe radiation” refer to collecting radiation of a probelight beam that has interacted with a sample. The probe light can becollected after reflection, scattering, transmission, evanescent wavecoupling, and/or transmission through an aperture probe.

“Confocal microscopy” refers to a form of optical microscopy in whichthe light collected at a detector is confined to light that passesthrough a small volume within the 3D focus volume of an opticalobjective on a sample. Confocal microscopy is often performed by placinga “confocal aperture” at a focal plane that is equivalent with the focalplane of the sample, thus blocking stray light that does not passthrough the focus volume on the sample.

A “detector” refers to a device that produces a signal indicative of thepower, intensity and/or energy of light/radiation incident on thedetector surface. The signal will generally be an electrical signal, forexample a voltage, current and/or an electrical charge. The detector maybe a photodiode, a phototransistor, a charge coupled device (CCD). Insome cases, a detector may be a semiconducting detector, for example asilicon PIN photodiode. A detector may also be an avalanche photodiode,a photomultiplier tube, or any other device that produce a change incurrent, voltage, charge, conductivity or similar upon incidence oflight. A detector may comprise a single element, multiple detectorelements, for example a bi-cell or quad-cell, a linear or twodimensional array of detector elements, including camera baseddetectors.

“Diffraction limit” of a light beam means the minimum separation of twooptical sources that can be distinguished by a detector. The Abbediffraction limit d for a microscope having a numerical aperture NA andoperating at a wavelength X is defined as d=λ/(2·NA). Physicalrestraints on the numerical aperture of a microscope prohibit very largenumerical apertures, and therefore the diffraction limit of a microscopedepends strongly upon the operating wavelength used for detection, withlarge wavelengths corresponding to relatively poor resolution and highwavelengths corresponding to increased precision.

“Direct light” and “surround light” both refer to light that issubstantially undeflected after interacting with a sample.

“Demodulate” or “demodulation” refers to extracting aninformation-bearing signal from an overall signal, usually, but notnecessarily at a specific frequency. For example, in this application,the collected probe light collected at a photo detector represents anoverall signal. The demodulation process picks out the portion that isbeing perturbed by infrared light absorbed by the sample. Demodulationcan be accomplished by a lock-in amplifier, a fast Fourier transform(FFT), a calculation of a discrete Fourier component at a desiredfrequency, a resonant amplifier, a narrow band bandpass filter, or anyother technique that largely enhances the signal of interest whilesuppressing background and noise signals that are not in sync with themodulation.

A “demodulator” refers to a device or system that performs demodulation.

“Figure of merit” refers to any metric or indicator of the relativequality of a signal or measurement. The figure of merit can for examplebe a measurement sensitivity, a signal strength, a noise level, a signalto noise ratio, a background level, a signal to background ratio, anycombination of these, or other metric that lets one rank the relativequality of a signal and/or measurement. Additionally, figures of meritrelevant to the embodiments described herein include image acquisitionrate, transverse resolution, temporal phase sensitivity, and spatialphase sensitivity.

“Focusing optic” refers to one or more optical elements with the abilityto focus light. A focusing optic can comprise one or more refractivelenses, curved mirrors, diffractive optics, Fresnel lenses, volumehologram, metamaterial, or any combination thereof or any other deviceor component capable of focusing radiation. “Collimating optic” refersto any of the above optical elements arranged in a way to generallycollimate radiation. In some cases the same optic(s) may serve as both afocusing optic and a collimating optic, for example focusing light inone direction of propagation and then recollimating the light in theopposite direction of propagation. In drawings herein are oftenillustrated for simplicity as a single simple lens. In practice they mayoften be groups of lenses. For example a microscope objective normallycomprising many lenses in a complex arrangement will just be indicatedby a single lens icon. Similarly, the use of a lens icon in a drawingdoes not imply that only a lens can be used to achieve the design goal.It is understood that any of alternate focusing optics defined above(e.g., curved mirrors, etc.) or any combination thereof can be used inplace of the simple lens shown in the drawings.

A “4f optical relay system” in the context of this application is anoptical system comprising at least two focusing optics and comprising anintermediate Fourier transform plane between two of the focusing optics.The simplest 4f relay system in this context can comprise two lensesspaced their focal lengths from the intermediate Fourier transformplane. The two lenses may have the same focal lengths in which case thesystem has unitary magnification, or the lenses may have different focallengths to enable additional magnification or demagnification in therelay system. The focusing elements need not be lenses and can insteadbe curved mirrors or any of the other optics as defined in the term“focusing optic.”

“Fluorescence” refers to the emission of light from a sample at onewavelength due to excitation at another wavelength due to fluorescentexcitation and emission processes.

“Illuminate,” “Illuminating,” and “Illumination” mean to directradiation at an object, for example a surface of a sample, the probetip, and/or the region of probe-sample interaction. Illumination mayinclude radiation in the infrared wavelength range, visible, and otherwavelengths from ultraviolet to a millimeter or more. Illumination mayinclude any arbitrary configuration of radiation sources, reflectingoptics, focusing optics, and any other beam steering or conditioningelements.

“Infrared absorption spectrum” refers to a spectrum that is proportionalto the wavelength dependence of the infrared absorption coefficient,absorbance, or similar indication of IR absorption properties of asample. An example of an infrared absorption spectrum is the absorptionmeasurement produced by a Fourier Transform Infrared spectrometer (FTIR)(i.e., an FTIR absorption spectrum). In general, infrared light willeither be absorbed (i.e., a part of the infrared absorption spectrum),transmitted (i.e., a part of the infrared transmission spectrum), orreflected. Reflected or transmitted spectra of a collected probe lightcan have a different intensity at each wavelength as compared to theintensity at that wavelength in the probe light source. IR measurementsare often plotted showing the amount of transmitted light as analternative to showing the amount of light absorbed. For the purposes ofthis definition, IR transmission spectra and IR absorption spectra areconsidered equivalent as the two data sets as there is a simplerelationship between the two measurements.

“Infrared source” and “source of infrared radiation” refer to one ormore optical sources that generates or emits radiation in the infraredwavelength range, generally at least a subset of the range between 2-25microns. The radiation source may be one of a large number of sources,including thermal or globar sources, supercontinuum laser sources,frequency combs, difference frequency generators, sum frequencygenerators, harmonic generators, optical parametric oscillators (OPOs),optical parametric generators (OPGs), quantum cascade lasers (QCLs),interband cavity lasers (ICLs), synchrotron infrared radiation sources,nanosecond, picosecond, femtosecond and attosecond laser systems, CO₂lasers, microscopic heaters, electrically or chemically generatedsparks, and/or any other source that produces emission of infraredradiation. The source emits infrared radiation in a preferredembodiment, but it can also emit in other wavelength ranges, for examplefrom ultraviolet to THz. The source may be narrowband, for example witha spectral width of <10 cm⁻¹ or <1 cm⁻¹ less, or may be broadband, forexample with a spectral width of >10 cm⁻¹, >100 cm⁻¹ or greater than 500cm⁻¹. Broadband sources can be made narrow band with filters,diffraction gratings, monochromators and other devices. The infraredsource can also be made up of one of discrete emission lines (e.g.,tuned to specific absorption bands of target species).

“Interacting” in the context of interacting with a sample means thatlight illuminating a sample is at least one of scattered, refracted,absorbed, retarded, aberrated, diverted, diffracted, transmitted, andreflected by, through and/or from the sample.

A “lock-in amplifier” is one example of a “demodulator” (defined above)and is a device, system, and/or an algorithm that demodulates theresponse of a system at one of more reference frequencies. Lock-inamplifiers may be electronic assemblies that comprise analogelectronics, digital electronics, and combinations of the two. They mayalso be computational algorithms implemented on digital electronicdevices like microprocessors, field programmable gate arrays (FPGAs),digital signal processors, single board computers, and personalcomputers. A lock-in amplifier can produce signals indicative of variousmetrics of an oscillatory system, including amplitude, phase, in phase(X) and quadrature (Y) components or any combination of the above. Thelock-in amplifier in this context can also produce such measurements atboth the reference frequencies, higher harmonics of the referencefrequencies, and/or sideband frequencies of the reference frequencies.

“Modulating” or “modulation” when referring to radiation incident on asample refers to changing the infrared laser intensity at a locationperiodically. Modulating the light beam intensity can be achieved bymeans of mechanical chopping of the beam, controlled laser pulsing,and/or deflecting the laser beam, for example by a tilting mirror thatis driven electrostatically, electromagnetically, with piezo actuatorsor other means to tilt or deform the mirror, or high speed rotatingmirror devices. Modulation can also be accomplished with devices thatprovide time varying transmission like acousto-optic modulators,electro-optic modulators, photo-elastic modulators, Pockels cells, andthe like. Modulation can also be accomplished with diffraction effects,for example by diffractive MEMS-based modulators, or by high-speedshutters, attenuators, or other mechanisms that change the intensity,angle, and/or phase of the laser intensity incident on the sample.

“Near infrared light” generally refers to a wavelength range of infrared(IR) light corresponding to 0.75-2 μm.

“Optical property” refers to an optical property of a sample, includingbut not limited to index of refraction, absorption coefficient,reflectivity, absorptivity, real and/or imaginary components of theindex refraction, real and/or imaginary components of the sampledielectric function and/or any property that is mathematically derivablefrom one or more of these optical properties.

“Optical response” refers to the result of interaction of radiation witha sample. The optical response is related to one or more opticalproperties defined above. The optical response can be an absorption ofradiation, a temperature increase, a thermal expansion, a photo-inducedforce, the reflection and/or scattering of light, change in brightness,intensity, optical phase, or other response of a material due to theinteraction with illuminating radiation.

A “narrowband light source” a light source with a narrow bandwidth orlinewidth, for example a light of linewidth smaller than 8 cm-1, but ingeneral it can be a light source with a linewidth narrow enough that thelinewidth does not cover a spectral range of interest of the sample.

“OPTIR” refers to Optical Photothermal Infrared Spectroscopy, atechnique in which a probe beam is used to measure the photothermaldistortion on a sample due to the absorption of infrared light. Theshorter wavelength of the probe beam provides spatial resolution muchhigher than can be achieved by conventional IR spectroscopy. The OPTIRtechnique generally produces at least one of infrared absorption spectraand/or infrared absorption images.

“Photothermal distortion” refers to a change in the properties of asample due to absorption of optical energy, for example the absorptionof IR radiation. The photothermal distortion may refer to a change inindex of refraction, reflectivity, thermal expansion, surfacedistortion, or other effects that can be detected with a probe beam. Aphotothermal distortion can impart a change in intensity, size,radiation distribution, direction, and/or optical phase of a probe beaminteracting with an IR absorbing region of a sample.

A “probe source,” “probe light source,” or “probe radiation source”refer to a radiation source that can be used for sensing of an opticalproperty of a sample. A probe light source can be used to sense theresponse of the sample to the incidence of light from the infrared lightsource. The radiation source may comprise a gas laser, a laser diode, adiode pumped solid state laser (DPSS), a superluminescent diode (SLD), anear infrared laser, a UV and/or visible laser beam generated via sumfrequency or difference frequency generation, for example. It may alsocomprise any or other sources of near-infrared, UV, and/or visible lightthat can be focused to a spot and/or imaged with a resolution on thescale smaller than 2.5 micrometer, and or even smaller than 1micrometer, and possibly smaller than 0.5 micrometer. In someembodiments, the probe light source may operate at a wavelength that isoutside the tuning or emission range of the infrared light source, butthe probe light source can also be a fixed wavelength source at a selectwavelength that does in fact overlap with the tuning range of theinfrared light source. A “probe light beam” or “sensing light beam” is abeam originally emitted from a probe light source.

“Probe beam” is a beam of light or radiation that is directed onto asample to detect a photothermal distortion or other optical changeresulting from the interaction of IR radiation with the sample, forexample to detect the absorption of IR radiation by the sample. Theprobe beam may be a tightly focused spot or may instead illuminate awide area of a sample.

“Raman” refers to light that is inelastically scattered from a sample atone or more wavelengths that are different from the excitationwavelength due to Raman scattering. “Raman spectroscopy” refers tomeasuring the spectroscopic content (Raman spectra) of Raman scatteredlight, for example the intensity of Raman scattered light as a functionof Raman shift. “Raman spectrometer” is a device for examining Ramanshifts in light collected from a sample and producing Raman spectraand/or Raman images.

“Scattered light” refers to light in which the propagation angle(s) ofthe light is altered due to interaction with a sample, such as bydiffraction. In the context of phase contrast microscopy, this may alsobe referred to as “diffracted light.”

“Signal indicative of” refers to a signal that is mathematically relatedto a property of interest. The signal may be an analog signal, a digitalsignal, and/or one or more numbers stored in a computer or other digitalelectronics. The signal may be a voltage, a current, or any other signalthat may be readily transduced and recorded. The signal may bemathematically identical to the property being measured, for exampleexplicitly an absolute phase signal or an absorption coefficient. It mayalso be a signal that is mathematically related to one or moreproperties of interest, for example including linear or other scaling,offsets, inversion, or even complex mathematical manipulations.

A “retarder” refers to an optical element that induces a relativeoptical phase delay in an optical path. Examples of retarders are waveplates, for example half wave plates, quarter wave plates and eight waveplates. One or more retarders/wave plates can be used to introduce anoptical phase difference between two polarizations of light, for exampleto introduce a phase difference between two paths of a quadratureinterferometer. A “variable retarder” is a retarder that can introducean optical phase delay that is controllable via an external signal, forexample a liquid crystal variable retarder.

A “spatial light modulator” is a device that provides positionaddressable control over the amplitude and/or optical phase of a lightbeam that is reflected off of it or transmitted through it. A spatiallight modulator can comprise a 2D array of electronically addressablevariable retarders, including liquid crystal variable retarders. Spatiallight modulators can also include reflective devices such as liquidcrystal on silicon (LCOS), and MEMS based devices like micro-mirrorarray devices.

“Spectrum” refers to a measurement of one or more properties of a sampleas a function of wavelength or equivalently (and more commonly) as afunction of wavenumber.

“Wide field” refers to using a camera or array detector to measure aplurality of sample locations substantially simultaneous, and not asingle point detector that measures a single point on a sample at atime. In other words, a wide field detection system looks at capturesentire frames or images corresponding to an extended region of a sample,rather than just data from a single point on a sample. A wide fieldregion may correspond to a region on a sample least 50 μm across, or atleast 100 μm, or at least 500 μm across.

The terms “about” or “approximate” and the like are synonymous and areused to indicate that the value modified by the term has an understoodrange associated with it, where the range can be ±20%, ±15%, ±10%, ±5%,or ±1%.

The term “substantially” is used to indicate that a result (e.g.,measurement value) is close to a targeted value, where close can mean,for example, the result is within 80% of the value, within 90% of thevalue, within 95% of the value, or within 99% of the value.

Embodiments described herein improve upon earlier photothermalcharacterization systems in that they provide more rapid samplecharacterization, eliminate artifacts endemic to QPI systems, and do notrequire burdensome sample preparation methods of conventional systems.Signal to noise can be enhanced along with optical efficiency whencompared to OPTIR and QPI systems that were previously state of the art,while expensive equipment such as high-speed cameras.

High-Performance Wide Field Photothermal IR Optical Phase Spectroscopy

FIG. 1 is a simplified schematic diagram of an embodiment of an OpticalPhotothermal Infrared (OPTIR) spectroscopy and imaging system for widefield chemical analysis using interferometric optical phasemeasurements. An infrared source 100 emits a beam of infrared radiation102 onto a region 108 of sample 110. IR beam 102 may be a beam directlyout of IR source 100, or may be optionally focused (or even expanded) byfocusing optic 104. In any case, the IR beam is arranged to illuminateda wide region of the sample, for example at least 25 μm across, butpreferably at least 50 μm across, or even >100 μm or >500 μm across,depending on the power level of the IR source and the desired size ofthe measurement region. When the wavelength of the infrared radiation102 is set to a wavelength corresponding to one or more IR absorptionbands of sample region 108, the absorbing regions heat up, causingphotothermal distortions in the IR absorbing regions of the sample.These photothermal distortions can comprise changes in thermalexpansion, deflection, deformation, size, shape, curvature,reflectivity, and/or index of refraction of the heated IR absorbingregions. These photothermal distortions can result in a change in theamplitude and/or optical phase of probe beam radiation that interactswith the sample that are measured to produce signals indicative of theIR absorption properties of the IR illuminated region of the sample.FIG. 1 illustrates one embodiment of extracting wide field measurementsof dynamic changes in optical phase resulting in localized sampleheating from IR absorption. These measurements in optical phase changecan be analyzed produce chemical images 148 and/or IR absorption spectra150.

To measure signals indicative of infrared absorption, probe beam 103from probe beam source 101 is transmitted through sample 110 at leastpartially overlapping IR illuminated region 108. Probe beam 103 is alsoarranged to illuminate a wide field region of the sample. Probe beam 103can come directly from probe beam source, for example as a collimatedlaser beam. It can also be focused or expanded as desired using focusingoptics not shown. In one embodiment, probe beam 103 can first be focusedonto the back focal plane of a condenser or objective to form acollimated illumination beam at the sample 110, for example as used inKohler illumination schemes. When the probe beam 103 passes through theIR illuminated region of sample 110, a pattern of the sample's opticalproperties is imprinted on the transmitted probe radiation 107 asillustrated schematically in INSET A. INSET A shows incident plane waves160 encountering materials 162 of different index of refraction thantheir surroundings resulting in distortions 164 in the transmittedwavefront. INSET A schematically illustrates retardations in the opticalphase of the transmitted plane waves passing through regions of higheroptical density (e.g., higher index of refraction). INSET A is aconceptual illustration and is not to scale or intended to correspond toactual data. INSET A illustrates a simple static case, but similarphysics applies in the case of a dynamic change in the optical phase dueto a photothermal distortion from IR absorption. The current embodimentenables rapid wide field measurement of subtle changes in optical phasedue to IR absorption of by regions of the sample, thus providingspatially resolved chemical analysis of the sample. Optical phase-basedmeasurement can be specifically advantageous because many samples, forexample biological materials, are highly transparent to visibleradiation. For this reason, they provide only very minor changes inintensity (amplitude) when light is transmitted through them. But eventhough biological materials can be highly transparent to visible light,they can still accumulate a large change in optical phase. For example,a typical biological cell can induce around 90° of optical phase changein transmission. For example, consider a biological cell that is 5 μmthick and with a typical index of refraction of 1.36 vs an index of1.335 for the surrounding aqueous media. This thickness and index changecause a retardation of 5 μm×(1.36-1.335)=0.125 μm, or around 0.23λ, orabout a quarter wavelength (˜90°). For biological samples measuringchanges photothermal changes in this relatively large optical phase canin some cases be more sensitive than measuring photothermal changes inrelatively small changes in optical intensity of highly transparentsamples.

To make a measurement of the sample imprint on the incident proberadiation 103, transmitted probe radiation 107 passing through sample110 is collected by focusing optic 109, typically a high numericalmicroscope objective, but can also be any other focusing optic. Thecollected probe radiation 111 is optionally reflected off mirror 112 (ordirected/steered by other optional optics not shown) to first focusingoptic 114, typically a tube lens of an optical microscope. Alternatelyfirst focusing optic 114 may be a separate optic for example mountedexternal to the body of an optical microscope. First focusing optic 114generally collimates the illuminating beam of probe radiationtransmitted through the sample. (Note that the light paths illustratedin FIG. 1 represent the paths of the illumination probe beam. FIG. 2A-Bseparately illustrates the paths of the illumination and imaging beams.)The generally collimated probe beam 115 then passes to a non-diffractivebeam splitter 116 that separates the transmitted, collimated probe beam115 into two separate beams 118 and 120, each of the beams 118 and 120corresponding to a different polarization of light. In the embodimentillustrated in FIG. 1 , the beam splitter 116 is illustrated as a Rochonprism (i.e., a prism that leaves one beam 118 undeflected and diverts asecond beam 120 by an angle θ). Alternate beam splitters may be used,for example a Wollaston prism that divides the two beams symmetricallyat ±0.

Both beams 118 and 120 are then incident on a second focusing optic 122,for example the first optic in a 4f relay system. Focusing optic 122focuses both the undeflected transmitted beam 124 and the deflected beam134. The focus 126 of beam 124 is arranged to pass through a spatialfilter 128. Spatial filter may for example be a small aperture pinhole,a clear region in a metal mask on glass, a pattern on a spatial lightmodulator or other device with a small transmissible aperture. As willbe explained in more detail associated with FIG. 2 , the aperture ofspatial filter 128 is chosen to essentially erase all imprint of thesample from transmitted probe beam that passes through the spatialfilter. This is done such that filtered beam 129 can act as anessentially feature-free reference beam against which angle deflectedbeam 134 will be interfered. Returning to beam splitter 116, in theembodiment shown this beam splitter divides the two beams bypolarization, such that one polarization is undeflected (beam 118) andthe beam 120 of orthogonal polarization is deflected by the angle θ. Themeans that beam 134 is nominally orthogonally polarized as compared tobeam 124. The current embodiment will re-interfere the undeflectedreference beam and deflected sample beam, so a polarization rotator 135is placed in the path of beam 134. Polarization rotator 135 for examplecan be a half wave plate which will rotate the polarization of beam 134by 90°. Alternately polarization rotator 135 can be formed by atransmissive spatial light modulator that can adjust the phase delay ofbeam 134. (Spatial filter 128 can also be formed using a spatial lightmodulator and/or a single spatial light modulator can perform both thespatial filtering and polarization rotating tasks.) Optional neutraldensity filter 136 can also be used to attenuate the probe beam 137 onthe sample path if desired such that it is similar or equal in intensityto the reference beam 129. Neutral density filter 136 may be a fixedattenuation or a variable attenuation, for example a variable neutraldensity filter wheel. Beam 137 that transmits through the polarizationrotator is then arranged to have essentially the same DC optical phaseas spatially filtered reference beam 129. Both beams then pass throughthird focusing optic 130 to be recombined at the surface 138 of detector132, typically a camera 132 or other array-based detector. The two beam131 and 139 that transmit through focusing optic 130 combine at surface138 of camera 132 to form an interferogram 140, comprising a largernumber of interference fringes 142 (two fringed indicated). A crosssection 144 through this interferogram 140 reveals an oscillatorypattern 146 with slight shifts in the positions of the peaks resultingfrom any phase lags resulting from optical retardation of objects on thesample in the path of the illuminating probe beam. (The drawing ofinterferograms 140/146 are simplified conceptual illustrations and notto scale.) Image interferograms 140 are analyzed by controller/analyzer152 that compares interferograms obtained with the IR light on (“hotframe”) to interferograms with the IR light off (“cold frame,” or atleast at a lower IR power level). Controller/analyzer 152 analyzes thedifferences in the interferograms between the hot and cold frames toproduce signals indicative of IR absorption, for example IR spectrum 150and/or IR absorption image 148. IR spectrum 150 is produced by plottingthe signal indicative of IR absorption analyzed from the hot/coldinterferograms as a function of the wavelength (or equivalentlywavenumber) of the IR source 100. IR absorption image 148 is created byplotting the signal indicative of IR absorption for one or morewavelengths of IR light over a plurality of locations of the sample,e.g. at a plurality of pixel positions on array sensor 132 and/or at aplurality of relative locations of IR/probe beam relative on sample 110,e.g. by translating the sample 110 under the probe and IR beams 102 and103. As described later, the analyzing of hot and cold frames can alsobe performed by an onboard controller/analyzer built into the arraysensor 132, e.g. on a field programmable gate array built into a camerasensor assembly.

The interference pattern 140 arises between beams that strike the camera138 arriving on two separate paths: (a) the deflected path that carriesthe optical imprint of the sample; (b) the undeflected path where thesample imprint has been erased by the spatial filter. The interferogrampattern 140 that appears on camera 132 has the series of linearinterference fringes 142 because sample and reference beams 139 and 131interfere with each other at the angle θ. The interferogram pattern 140may have the general form of:

I(x,y)=I _(r)(x,y)+I _(s)(x,y)+√{square root over (I _(r)(x,y)I_(s)(x,y))} cos(2k×sin θ+ϕ(x,y))  Eq. 1:

Where I(x,y) is the intensity measured at x,y locations of the camerasensor, I_(r) is the reference field intensity, I_(s) is the samplefield intensity, k is the wavevector i.e. k=2π/λ, and ϕ(x,y) is thelocal optical phase difference between the sample and reference path,including any phase differences introduced by the transmission of lightthrough the sample.

The period Δx of the interference fringes can be estimated by theequation:

$\begin{matrix}{{\Delta x} = \frac{\lambda}{2\sin\theta}} & {{Eq}.2}\end{matrix}$

The interferogram images can be analyzed as described in a followingsection entitled “Phase reconstruction and differential phasecalculation” to create a signal indicative of IR absorption by thesample over a wide area.

Table 1 below estimates the performance that can be achieved with theembodiment of FIG. 1 as compared to the performance estimated from theapparatus of Tamamitsu et al, as best discerned from the publication MiuTamamitsu, Keiichiro Toda, Ryoichi Horisaki, and Takuro Ideguchi,“Quantitative phase imaging with molecular vibrational sensitivity,”Opt. Lett. 44, 3729-3732 (2019), https://doi.org/10.1364/OL.44.003729.The table inputs for the Tamamitsu publication either come from thepublication directly or from manufacturer's specifications forcomponents identified. The bottom line performance factor indicated inthe number of photons that arrive at each camera pixel per second. Ascan be seen by TABLE 1, the embodiment of FIG. 1 can achieve more thanthree orders of magnitude more photons per second. For a well optimizedoptical system the SNR scales like the square root of the photon flux.Thus, the current embodiment provides and improvement of

$\sqrt{\frac{{4.26E} + 08}{{1.76E} + 05}} = {49X}$

in SNR over the estimated performance of the Tamamitsu publication.

TABLE 1 Current Tamamitsu Embodiment (estimate) Wavelength 532 nm 517 nmenergy per photon 3.74E−19 J 3.84E−19 J input laser power 200 mW 1500 mWrep rate 50000 Hz 1000 Hz rep period 20 psec 1 msec visible pulse 1 psec130 nsec time duty cycle  5% 0.01%   output laser 0.01 0.000195 powerenergy per pulse 0.2 uJ 0.195 uJ photons per 5.35E+11 5.07E+11 pulseinput beam 1 mm 2 × 4 mm diameter tube lens 40 40 magnificationMagnified beam 40 mm 120 mm diameter at camera photon areal 4.26E+14photons/ 4.48E+13 photons/ flux m² m² pixel size 5 μm 3.45 μm photonsper 10600 photons 534 photons pixel per pulse camera frame 3300 fps 60fps rate optical efficiency 80% 33% camera 300 μsec 17 msec sec exposuretime pulses per 15 16 exposure total photons 130,000 2,900 per exposureTotal photons 4.26E+08 1.76E+05 per second Max estimated 20,600 420 SNRin 1 sec

The following is a summary of some key factors supporting the higherphoton flux of the current embodiment. In one embodiment, the probesource is a diode pumped solid state laser, 532 nm wavelength with atleast 200 mW of optical power. Such lasers are available for examplefrom Cobolt (Hubner) and Coherent. The lasers may be continuous wave(CW) or pulsed. In the case of CW laser, a modulator may be used to gatethe probe pulses to be at a desired delay time after the start of the IRpulses. While the probe source used in Tamamitsu had a pulse limit of130 nsec, the use of a CW laser with an electro-optic modulator providesessentially unlimited pulse duration up to the repetition rate of the IRsource. For the table above, an IR pulse rep rate of 50 kHz was chosen,though rates up to a few MHz are also available, for example usingquantum cascade laser sources from Daylight Solutions or BlockEngineering. Suitable electro-optic modulators, such as Pockels cellsand drive electronics are available from vendors like Eksma Optics,ConOptics, G&H and others. A significant advantage of the use of a diodepumped solid state laser with a Pockels cell is that this arrangementcan achieve very small focused spots with high optical throughput. DPSSlasers from Cobolt for example have a small round beam and laser beamquality factor M² of less than 1.1, compared to elliptical beamsproduced by many diode lasers. This allows much more efficient opticalcoupling for example through spatial filters and through relay optics.Another critical factor is providing enough photons at the camera to beabove the dark current noise and pixel shot noise. For detection ofsmall changes like the photothermal modulations discussed herein, it isdesirable to have enough light per exposure to work near the saturationlimit of the camera. Even with a frame rate of around 60 frames persecond, the Tamamitsu approach is estimated to have of order 3000photons per exposure. The noise goes like the square root of the numberof photons, so this would provide a best case single frame SNR of

$\frac{3000}{\sqrt{3000}} = 54.$

By comparison, me current embodiment could achieve a single frame SNR ashigh as

$\frac{130000}{\sqrt{130000}} = 360.$

Additionally, the current embodiment can capture many more frames persecond. In one second, the current embodiment can co-average 3300 ormore frames, leading to a further SNR improvement of 57×, for an SNR inone second of 57×360=20,600. By comparison, the Tamamitsu limit of 60frames per second only provides ˜7.7×SNR improvement or an overall SNRof 54×7.7=420. In practice, however, the Tamamitsu paper reported outsignificantly worse results than this, achieving a SNR of around 5 fortheir final photothermal detection sensitivity in a 1 second exposure.Two key factors that also contribute to the ability of the currentembodiment of achieving much higher SNR are the ability to use highframe rate cameras based on high optical throughput and the ability toperform high speed calculations of the photothermal phase change. Bothof these will be discussed later in this specification.

FIG. 2 illustrates a portion of the optical paths of FIG. 1 in moredetail. Specifically, FIGS. 2A and 2B illustrate side by side the lightpaths for the illumination beams (FIG. 2A) and the imaging beam (FIG.2B). FIG. 2 is arranged with the optical path in a linear configuration,i.e. oriented vertically for simplicity, i.e. omitting fold mirror 112of FIG. 1 . It should be understood that, in practice, the opticalarrangement can be folded in two or three dimensions for a more compactarrangement if desired. Starting with the illumination path in FIG. 2A,an illumination beam 200 is used to illuminate a region 201 on sample202. Illumination beam 200 can in some embodiments be substantiallycollimated and illuminate a wide area of sample 202, for example aregion >25 microns, across, >50 microns across, 100 microns across, >500microns across or even >1000 microns across. The illumination beam isarranged to at least partially overlap with IR beam 204, for exampleemitted by IR source 100 of FIG. 1 . IR beam 204 is used to exciteresonances in the sample, for example associated with molecularvibrations. Illumination probe light 200 passing through sample 202 canbe collected by focusing optic 206, e.g. a microscope objective, andfocused to a point 208 resulting in an expanding beam 210 that strikesfocusing optic 212, for example a microscope tube lens. Focus point 208can be arranged to be at the focal distance of focusing optic 212 suchthat a collimated illumination beam 214 emerges downstream of focusingoptic 212.

The re-collimated illumination beam 214 can be directed toanon-diffractive beam splitting element 216, for example a beamsplitting prism like a Wollaston or Rochon prism. The center of the beamsplitting element 216 can also be arranged to be at a conjugate focalplane of the sample 202 such that an image of the sample 215 issuperimposed on the beam splitting element 216. (This will be describedin more detail associated with FIG. 2B.) Beam splitting element 216divides the illumination beam onto two paths 218 and 220, separated byan angle θ. In the example shown, one of the beams 218 is undeviated andthe other beam 220 is deviated by the angle θ, as would be the case forusing a Rochon prism. Alternately, a Wollaston prism can be used, whichwill deviate both beams by ±θ. In the case of a Rochon or Wollaston orsimilar polarization sensitive beam splitter, the two beams that emergewill have substantially orthogonal polarization. Both beams 218 and 220are directed towards focusing optic 222, typically the first lens in a4f relay system. Focusing optic 222 refocuses the transmitted beams 224and 240 to focused points 226 and 242 respectively. At the focus of oneof the beams a spatial filter 228 is placed. The spatial filter is sizedto essentially erase all information about the sample from the beam,producing an essentially featureless plane wave beam 230 that will forma reference beam for the interference. The other beam 242 optionallypasses through a polarization rotator 244, for example a half waveplate, return transmitted beam 246 to a polarization that is matchedwith reference beam 230 such that they will interfere. Both beams 230and 246 are directed through a final focusing optic 232, e.g. the 2^(nd)lens of a 4f relay system. Focusing optic 232 recollimates theillumination beams into beams 234 and 248. As mentioned previously,reference beam 234 is an essentially featureless plane wave while beam238 carries an imprint of the sample. Both beams are interfered at anangle (e.g. the angle θ) to produce an image interferogram at thesurface 236 of wide area detector 238, typically a camera or other arraysensor.

FIG. 2B illustrates the optical path of the imaging beams using the sameoptical layout of FIG. 2A. In this case, consider the light that isscattered from a single image point 249 on sample 202. The scatteredlight 250 emerging from a single image point 249 on sample 202 iscollected by the same focusing optic 206 described in FIG. 2A, e.g. amicroscope objective. Since the sample is typically placed at the focaldistance of focusing optic 206, the imaging beam 251 emerging fromfocusing optic 206 will be substantially collimated. The collimated beam251 is directed to focusing optic 212 (e.g. a microscope tube lens)which then refocuses the transmitted beam 252 to a point 253. And inturn each other image point in sample 202 that is within the field ofview of the objective 206 will be focused to a corresponding point bytube lens 212 creating a magnified image 215 at the tube lens focus. Asdescribed above associated with FIG. 2A, the non-diffractive beamsplitting element 216 is placed at or near this focus to create twoemerging beams 254 and 256 separated by an angle θ. One of the beams, inthis case beam 258 is arranged to strike a spatial filter 228 with asmall aperture. The spatial filter 228 will typically have a small hole,e.g. of order25 microns, thus permitting almost none of the imaging beamto pass through, illustrated by the absence of an imaging beam past thespatial filter 228. (The details of the sizing of the spatial filterdepend on the size of the input probe beam and the focal lengths of thefocusing optics used, including for example the magnification created bythe objective, tube lens and the 4f optical system.) Note that somelight does in fact pass through this spatial filter, as illustrated inFIG. 2A, but only a portion of the undeviated illumination light,whereas the vast majority of the imaging/scattered light is blocked. Onthe other hand, the deflected beam 260 transits on a path with nospatial filter, thus preserving the image information from the scatteredlight on this second path. Beam 260 optionally passes through apolarization rotator 244 and neutral density filter 245 as describedwith FIG. 2A. The image beam 262 is then refocused by focusing optic 232onto the surface 236 of wide area detector 238, e.g. at the surface of acamera. The interference of the imaging beam 264 with reference beam 234of FIG. 2A and with the illumination beam 248 creates an interferogramon the surface 236 of the camera 238. In the absence of any scatteringby the sample, there will be no imaging beam 264 and the resultinginterferogram will comprise essentially parallel lines with consistentfringe spacing. In the case that light is scattered by the sample, beams248 and/or 264 will carry imprints of the sample that will result in adeviation of the interference fringes. These deviations can be analyzedto make an optical phase map of the sample. Further, these phase mapscan be made with and without IR illumination, for example by pulsing orotherwise modulating IR beam 204 that at least partially overlaps withthe illumination beam 200 and imaging beam 250 and analysis of shifts inthe fringe pattern can be used to determine a signal indicative of theIR absorption by the sample. Note that in FIGS. 1, 2A and 2B, the sampleand reference paths can also be reversed, e.g. such that the deflectedbeam passes through the spatial filter and becomes the reference beam.In some embodiments, it can be preferential that both beams are equallydeflected in opposite directions, as in the case of a Wollaston prism.This can be advantageous as it ensures that both sample and referencebeams are substantially the same path length, which can be desirablewhen using an illumination source with low coherence length. Additionalcompensation optics (not shown) can also be placed in the beam path toaccount for any phase differences associated with items in one path,e.g. the polarization rotator 244 and neutral density filter 245. Thesecan be for example pieces of glass with thickness and index chosen toprovide a similar optical path length difference as any opticalcomponents placed in the other path.

Phase Reconstruction and Differential Phase Calculation

There are a variety of ways to calculate the local phase from thisinterferogram and several have been described in the literature ofquantitative phase imaging (QPI), including the use of Fouriertransforms (Mitsuo Takeda, Hideki Ina, and Seiji Kobayashi,“Fourier-transform method of fringe-pattern analysis for computer-basedtopography and interferometry,” J. Opt. Soc. Am. 72, 156-160 (1982),https://doi.org/10.1364/JOSA.72.000156), a Hilbert transform (TakahiroIkeda, Gabriel Popescu, Ramachandra R. Dasari, and Michael S. Feld,“Hilbert phase microscopy for investigating fast dynamics in transparentsystems,” Opt. Lett. 30, 1165-1167 (2005),https://doi.org/10.1364/OL.30.001165, and U.S. Pat. No. 8,772,693), andderivative methods (Basanta Bhaduri and Gabriel Popescu, “Derivativemethod for phase retrieval in off-axis quantitative phase imaging,” Opt.Lett. 37, 1868-1870 (2012) https://doi.org/10.1364/OL.37.001868), eachincorporated by reference.

In the case of combining infrared spectroscopy with a quantitative phaseimaging (QPI) technique, it is desirable to rapidly calculate thedifference in the measured phase with and without the presence of IRlight. For example two images can be obtained, one with the IR light onand another with the IR light off. Then both images can be analyzed toreconstruct two phase images with and without IR light. Then the twoimages are subtracted and the difference in the two phase images isindicative of the IR absorption by the sample. To achieve high signal tonoise and/or high measurement throughput, it can be desirable to usecameras or other sensor arrays that can support high frame rates, forexample >1000 frames/second, >10,000 frames per second or even >100,000frames per second. Some of the approaches used within the QPI communityfor computing local phase from the image interferograms can becomputationally intensive and may be challenging to implement at highcamera frame rates that are desirable for wide field OPTIR techniques. Ahighly efficient technique is outlined in a following section thatsupports rapid measurements of the phase difference induced with IRabsorption by the sample.

First, consider Eq 1: rewritten here for a single x,y point:

I=I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(2k×sinθ+ϕ)  Eq. 3:

Eq. 3 suggests a generally oscillatory waveform via the term cos(2 k×sinθ) accompanied by a DC offset set by I_(r)+I_(s). The optical phase ϕintroduced by the sample causes variations in the period of theoscillatory behavior of the interferogram. Below is described anefficient method to extract both the DC offset and the optical phase ϕ.

FIG. 3 illustrates a method of rapidly calculating the optical phasefrom an interferogram. FIG. 3A illustrates a portion of a row 300 ofsensors in a sensor array, e.g. pixels in a camera based detector.Example pixels are labeled 301, 302, 303, and 304. FIGS. 3B and 3Cillustrate cross-sections of interferograms 305 b and 305 c that areincident on different rows the sensor array surface, e.g. on differentrows on a camera sensor chip. In practice, the sensory array may havehundreds or even thousands of pixels in each row and column. Theinterferograms 305 b and 305 c may have different relative phaserelationships to the grid of camera pixels, for example due to opticalphase differences in the regions imaged to the different rows. It isthen the object to calculate the relative phase differences from thepixel intensities. In one embodiment, the spacing of the pixels and theangle of the interferogram is arranged such that there is a differenceof roughly 90° between adjacent pixels. Alternately, it can be arrangedto have a 90° phase difference over N pixels, where N is an integer.E.g. bins of multiple pixels can be selected with an average phasedifference of 90° between bins. The 90° phase difference being referredto here is not the optical phase change due to the sample, but ratherthe phase that is accumulated in the lateral direction due to thereference and sample waves interfering at an angle, i.e. the (2k×sin θ)term in the cosine of Eq. 3. So specifically, the optical system isarranged such that:

$\begin{matrix}{{2{kN}\Delta x\sin\theta} = \frac{\pi}{2}} & {{Eq}.4}\end{matrix}$

where Δx is the distance between pixels, i.e. the pixel size. Thiscondition can be met by arranging suitable selection of the camera pixelsize, magnification of the 4f system and tube lens, wavelength, andinterfering beam angle θ. FIG. 3 illustrates the condition where N=1,i.e. each camera pixel advances the phase of the 2k×sin θ term by 90°(π/2). The intensity of light from the interferogram captured by thecamera pixels 301-304 is indicated by values I₁, I₂, I₃ and I₄. Thesevalues will be used to calculate the optical phase ϕ. The optical systemis also arranged with sufficient magnification such that there aremultiple camera pixels per resolution element in the microscope. In thiscase the optical phase ϕ has a roughly constant value over a number ofadjacent pixels as do the sample and reference intensities I_(r) andI_(s). Specifically, it is assumed that these values are roughlyconstant for at least pixels 301-303, or alternately 301-304. In thiscase, equations for the intensities I₁, I₂, I₃ and I₄ can be written asfollows:

I ₁ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(ϕ)  Eq: 5

I ₂ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(90°+ϕ)  Eq:6

I ₃ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(180°+ϕ)  Eq.7:

I ₄ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(270°+ϕ)  Eq.8:

Equation 5 was written assuming x=0, and then Eqs. 6-8 advance the phaseof the 2 k×sin θ term in Eq. 3 by a 90° (π/2) increment for each pixel.Using trigonometric identities, these equations can be rewritten:

I ₁ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(ϕ)  Eq. 9:

I ₂ =I _(r) +I _(s)−√{square root over (I _(r) I _(s))} sin(ϕ)  Eq. 10:

I ₃ =I _(r) +I _(s)−√{square root over (I _(r) I _(s))} cos(ϕ)  Eq. 11:

I ₄ =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} sin(ϕ)  Eq. 12:

Three or more of these equations can be combined to solve for theoptical phase ϕ. For example, adding Eqs. 9 and 11 results in:

I ₁ +I ₃=2(I _(r) +I _(s))  Eq. 13:

And then:

$\begin{matrix}{( {I_{r} + I_{s}} ) = \frac{I_{1} + I_{3}}{2}} & {{Eq}.14}\end{matrix}$

Rearranging Eqs. 9 and 10 give

$\begin{matrix}{{\cos(\phi)} = \frac{I_{1} - ( {I_{r} + I_{s}} )}{\sqrt{I_{r}I_{s}}}} & {{Eq}.15}\end{matrix}$ $\begin{matrix}{{\sin(\phi)} = {- \frac{I_{2} - ( {I_{r} + I_{s}} )}{\sqrt{I_{r}I_{s}}}}} & {{Eq}.16}\end{matrix}$

Eq. 16 can be divided by Eq. 17 to get:

$\begin{matrix}{{\tan(\phi)} = {- \frac{I_{2} - ( {I_{r} + I_{s}} )}{I_{1} - ( {I_{r} + I_{s}} )}}} & {{Eq}.17}\end{matrix}$

Plugging in Eq. 14 into Eq. 17 gives:

$\begin{matrix}{{\tan(\phi)} = {{- \frac{I_{2} - ( \frac{I_{1} + I_{3}}{2} )}{I_{1} - ( \frac{I_{1} + I_{3}}{2} )}} = {- \frac{{2I_{2}} - I_{1} - I_{3}}{I_{1} - I_{3}}}}} & {{Eq}.18}\end{matrix}$

Eq. 18 can then be solved for the phase ϕ:

ϕ=atan 2(2I ₂ −I ₁ −I ₃ ,I ₃ −I ₁),  Eq. 19:

where atan 2 is the two-argument inverse tangent. Other forms of theinverse tangent may also be used. Phase unwrapping techniques can beapplied to remove any discontinuities in the phase. Note that Eq. 19calculates a signal indicative of the phase with as few as 3-pixelintensity values and thus can be computed very rapidly. More noiserejection can be achieved by using more pixels, for example binningpixels vertically (in the y direction). More accurate measurements ofthe DC offset (I_(r)+I_(s)) can also be obtained by using for exampleother combinations of Eq. 9-12, for example using the sum of Eqs. 10 and12 in addition to Eqs. 9 and 11. As mentioned previously, it is alsopossible to have the phase increment between pixels to be less than 90°to be able to bin pixels in the X direction for measurements of theI₁-I₄ values.

A differential photothermal signal can be constructed by measuring thechange in the phase Δϕ with the IR light on vs. off. That is:

Δϕ=ϕ_(IR on)−ϕ_(IR off)  Eq. 20:

This quantity Δϕ is then indicative of the change in optical phaseresulting from IR absorption by the sample. The quantity Δϕ can then beplotted as a function of position for one or more IR excitationwavelengths to produce a map indicative of the distribution of differentchemical species. The quantity Δϕ can also be plotted as a function ofdifferent excitation wavelengths (or equivalently wavenumber) to producea signal indicative of the IR absorption properties of a sample, forexample an infrared absorption spectrum.

FIG. 4 shows an illustration of results calculated using the phasecalculation described above. Data was simulated using one pixel per 90°of phase shift per camera pixel from the 2 k× sin θ term of Eq. 3. Plot400 shows the intensities 402, 404 and 406 of adjacent pixels with the90° phase shift while varying the input optical phase. (As describedearlier, instead of individual pixels, multiple pixels can be binnedtogether in the X and or Y directions as long as the two sets of pixelsthat are binned together in the X axis an average phase difference of90° in the 2 k× sin θ term of Eq. 3.) As can be seen this arrangementcauses traces 402, 404 and 406 have a quadrature relationship to eachother (i.e., these two traces are also 90° out of phase). Plot 408 showsthe reconstruction of the phase ϕ from the three intensity values foreach input optical phase in plot 400, using Eq. 19 above. Trace 410shows a substantially accurate reconstruction of the input phase. Thissimulation was performed with a peak-to-peak noise amplitude of 4% foreach camera pixel. Improvements in SNR can be achieved by co-averagingthe results of multiple camera frames and/or by binning more pixels asdescribed above.

The following section outlines another way to extract the signalindicative of IR absorption. In this case it is assumed that at anypoint on the sample the optical phase has a DC value ϕ₀ that isperturbed by IR absorption changing the DC phase by a small increment δ.That is:

ϕ=ϕ₀+δ, where δ is small.  Eq. 21:

Plugging this into the cos(2k×sin θ+ϕ) term of Eq. 3 results in:

cos(2k×sin θ+ϕ₀+δ)  Eq. 22:

Next, the compound angle formula is applied cos(A+B)=cos A cos B−sin Asin B, where A=2k×sin θ+ϕ₀ and B=δ. This results in:

cos(2k×sin θ+ϕ₀+δ)=cos(2k×sin θ+ϕ₀)cos δ−sin(2k×sin θ+ϕ₀)sin δ  Eq. 23:

Using small angle expansions cos δ≈1 and sin δ≈δ, Eq. 23 can berewritten as:

cos(2k×sin θ+ϕ₀+δ)=cos(2k×sin θ+ϕ₀)−sin(2k×sin θ+ϕ₀)δ  Eq. 24:

This in turn can be solved for δ to give:

$\begin{matrix}{\delta = \frac{{\cos( {{2{kx}\sin\theta} + \phi_{0} + \delta} )} - {\cos( {{2{kx}\sin\theta} + \phi_{0}} )}}{- {\sin( {{2{kx}\sin\theta} + \phi_{0}} )}}} & {{Eq}.25}\end{matrix}$

Now consider the intensities of the camera pixels of FIG. 3A in theconditions of the IR light on (“hot”, subscript h) and with the IR lightoff (“cold”, subscript c), using Eqs. 9-12 starting with x=0.

I _(1h) =I _(r) +I _(s)+√{square root over (I _(r) I _(s))}cos(ϕ₀+δ)  Eq. 26:

I _(1c) =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} cos(ϕ₀)  Eq.27:

I _(2c) =I _(r) +I _(s)+√{square root over (I _(r) I _(s))} sin(ϕ₀)  Eq.28:

I _(3c) =I _(r) +I _(s)−√{square root over (I _(r) I _(s))} cos(ϕ₀)  Eq.29:

I _(4c) =I _(r) +I _(s)−√{square root over (I _(r) I _(s))} sin(ϕ₀)  Eq.30:

Subtracting Eq: 27 from Eq 26 results in:

I _(1h) −I _(1c)=√{square root over (I _(d) I_(s))}(cos(ϕ₀+δ)−cos(ϕ₀)),  Eq. 31:

which is proportional to the numerator in Eq. 25. Subtracting Eq. 30from Eq. 28 gives:

I _(2c) −I _(4c)=2√{square root over (I _(r) I _(s))} sin(ϕ₀),  Eq. 32:

which is proportional to the denominator in Eq. 25.Dividing Eq. 31 by Eq. 32 gives:

$\begin{matrix}{\frac{I_{1h} - I_{1c}}{I_{2c} - I_{4c}} = {\frac{\sqrt{I_{r}I_{s}}( {{\cos( {\phi_{0} + \delta} )} - {\cos( \phi_{0} )}} )}{2\sqrt{I_{r}I_{s}}{\sin( \phi_{0} )}} = \frac{( {{\cos( {\phi_{0} + \delta} )} - {\cos( \phi_{0} )}} )}{2{\sin( \phi_{0} )}}}} & {{Eq}.33}\end{matrix}$

Which is the same as Eq. 25 except for a factor of 2 and a minus sign.Making these adjustments gives:

$\begin{matrix}{\delta = {2\frac{I_{1h} - I_{1c}}{I_{4c} - I_{2c}}}} & {{Eq}.34}\end{matrix}$

Equation 34 shows how a signal indicative of IR absorption δ can becalculated extremely rapidly using only the intensities of nearby pixelsunder the hot/cold state and with extremely simple computation. Thisapproach provides the means to use sensitive interferometric techniquesthat provide in fact a quantitative measurement of the differentialoptical phase change due to IR absorption, but without the need toperform a separate quantitative measurement of the DC optical phase, atask that can be computationally intensive. The approach leading to Eq.34 also eliminates the need to be concerned about phase discontinuitiesor apply phase unwrapping techniques. This simplification occurs becauseof the use of the small angle approximation that the differential phasechange δ is small. This approximation, however, is justified in almostall cases because of the nature of the photothermal effect. Typicalmaterials introduce a change in index of refraction of around 10⁻⁴/° C.of temperature change. With a sample temperature increase of even 10°C., the max sample index change is around 10⁻³. The phase change is alsocommensurately small. Consider the previous example of a biological cellwith an optical path change of around 0.125 um, resulting in a DC phasechange of ˜90° or π/2. If the entire cell absorbed IR light and heatedup by 10° C., the resulting change in optical phase would be aroundπ/2×1E-3=0.001570796. The small angle approximation is appropriate sincesin(0.001570796)=0.001570796, i.e. sin δ=δ to very high accuracy in thiscase. For thinner samples, sub-cellular components, and/or smallertemperature rises (desirable for biological samples), the differentialphase change will be even smaller. So in almost cases the small angleapproximation is appropriate and Eq. 34 is applicable. Note that otherformulations of pixel intensities can also be used, for example binningmultiple pixels in the X and or Y directions as described previously.Signal to noise can be improved by coadding/coaveraging multiple cameraframes and/or multiple calculations of Eq. 34. Note also that Eq. 30contains the same cosine term as Eq. 27, so the I_(3h) and/or I_(3c)terms can be use in addition to or in place of the I_(1h) and I_(1c)terms of Eq. 34.

The approach described above can also apply if instead of measuringadjacent pixels with 90° incremental phase shifts, the intensities canbe measured at the same pixel but at successive optical pathdifferences, for example at three optical phases 90 degrees apart. Forexample, a transmissive variable phase retarder can be included in thepath of one or more of the reference beam and sample beam to introducesuccessive phase shifts. Suitable variable retarders are sold forexample by Thorlabs, Edmund Optics, Meadowlark Optics and others. Forexample, one hot frame and one cold frame can be measured with 0 degreesphase shift to obtain the intensities of Eq. 26 and 27 and then two coldframes at 90° and 270° can be measured to obtain the intensities of Eqs.30 and 31. Then these intensities can be combined to calculate thedifferential phase according to Eq. 34. This approach avoids the need toarrange a specific phase relationship between adjacent pixels.

The signals indicative of IR absorption δ can be calculated extremelyquickly for example using Eq. 34, because of the computationalsimplicity. This efficient computation is critical to enabling highcamera frame rates and high signal to noise ratios. More specifically,for continuous operation, the practical camera frame rate is constrainedby how quickly the accompanying calculation of differential photothermalphase change δ can be performed. The embodiments described herein canachieve calculation efficiencies sufficient to permit camera frame ratesin excess of 100 frames per second (fps), >1,000 fps or even >10,000fps. The table below summarizes benchmark computation times and enabledframe rates for the computation of Eq. 34 vs. other computationalgorithms common in quantitative phase imaging as described in the QPIliterature, for example the Hilbert Transform and Fast FourierTransforms (FFT), as described in Mitsuo Takeda, Hideki Ina, and SeijiKobayashi, “Fourier-transform method of fringe-pattern analysis forcomputer-based topography and interferometry,” J. Opt. Soc. Am. 72,156-160 (1982), https://doi.org/10.1364/JOSA.72.000156), and TakahiroIkeda, Gabriel Popescu, Ramachandra R. Dasari, and Michael S. Feld,“Hilbert phase microscopy for investigating fast dynamics in transparentsystems,” Opt. Lett. 30, 1165-1167 (2005),https://doi.org/10.1364/OL.30.001165, and U.S. Pat. No. 8,772,693.

Benchmark calculations were performed with the different algorithmsusing LabVIEW on a desktop computer using a Intel Xeon CPU ES-1607 v2running at 3.00 GHz using 512×512 pixels. The results are shown in thetable below.

512 × 512 pixels Calculation Equivalent Algorithm time frame rate FFT22.9 msec 44 fps Hilbert 15.2 msec 66 fps Eq. 34 1.4 msec 714 fps

It is apparent that the computational simplicity of Eq. 34 enables muchshorter computation times and much higher frame rates. If a smallernumber of pixels are uses, even higher frame rates can be achieved. Forexample, using 128×128 pixels, the computation time for Eq. 34 is 0.03msec, providing an equivalent frame rate up to 33,333 fps. The fastercomputation time and faster frame rates have a significant impact onsignal to noise ratio. For example, consider a one second acquisitiontime, where the Hilbert transform would support a maximum of 66 cameraframes acquired, whereas Eq. 34 would enable 714 frames. The SNRgenerally improves with the square root of the number of co-averaged orco-added camera frames. While the Hilbert transform would only supportan SNR improvement of √{square root over (66)}=8.1, Eq. 34 provides anSNR improvement of √{square root over (714)}=26.7. Using 128×128 pixelswhich enables the 33,333 fps, provides an SNR improvement of √{squareroot over (33,333)}=182. These high frame rates are also enabled bysignificantly higher optical throughput of the current embodiments, asdescribed later.

Note that the calculation times in the table above can also be improveddramatically using a dedicated embedded processor, for example a fieldprogrammable gate array (FPGA) which can perform many pixel calculationsin parallel. Camera sensor systems can be purchased or assembled withon-board FPGAs. For example the IL5 high speed camera from Fastec has anonboard FPGA that can be programmed to perform calculations like Eq. 34and supports a camera frame rates of 3300 fps at 640×480 pixels and 6300fps at 320×230 pixels. The MEMREMCAM HX-7s by nac Image Technologysupports frame rates as high as 12,000 fps for 640×480 pixels.

FIG. 5 shows an alternate embodiment of a wide field setup for OPTIRoptical phase measurements. FIG. 5 is based on FIG. 1 and whereidentical numerical callouts are used the associated descriptions fromFIG. 1 apply as appropriate. FIG. 5 shows the use of a non-common pathMach-Zender interferometer for wide field measurements of IR absorptionby a sample. Mach-Zender approaches have been used for Quantative PhaseImaging as described for example by Christopher J. Mann, Lingfeng Yu,Chun-Min Lo, and Myung K. Kim, “High-resolution quantitativephase-contrast microscopy by digital holography,” Opt. Express 13,8693-8698 (2005), hereby incorporated by reference. In the embodiment ofFIG. 5 , probe beam 103 from probe beam source 101 is divided on twopaths by beam splitter 500. One path 502 passes through the sample andobjective as described with FIG. 1 . A second portion of the beam thatwill serve as a reference beam is directed on an alternate path 504where the beam is optionally turned by one or more mirrors 506. Thereference beam is also optionally magnified and recollimated by focusingoptics 506 and 508 (or any equivalent beam expansion scheme) such thatthe beam diameter is similar to that of collimated probe beam 115. Thereference beam may also optionally pass through a spatial filter 507 toensure that the reference beam is an essentially a featureless referencebeam. The reference beam can optionally passes through a variable phaseretarder 510 to adjust the relative phase of the reference beam relativeto the sample beam. One or more of the sample and reference beam pathsmay also include a variable attenuator or neutral density filter toadjust the relative intensities on one or both arms. The reference beam512 is then recombined with sample beam 514 at beam combiner 516. (Beamcombiner 516 is generally just a beam splitter used in reverse.) Therecombined sample and reference beams then interfere at the surface 138of array detector (e.g., camera) 132. Note that the illumination beamsare drawn in this case and that the sample image beam paths are morelike FIG. 2B. The combination of the sample and reference beams at arraydetector 132 causes an interference pattern spread over the pixels ofthe detector. In the case that the two beams are collinear, there willbe a roughly constant phase on a featureless sample. On a sample withscattering/phase retarding objects, interference patterns will form onthe sample indicative of the optical phase difference between the sampleand reference paths, i.e. capturing an imprint of the phase distortionsintroduced by the sample. In the case that the sample and referencebeams are interfered at a small angle, an oscillating interferogramsimilar to 146 in FIG. 1 will be superimposed on the sample inducedphase distortions. In either case, the phase reconstruction processesdescribed above can be applied. In the case that the sample andreference beams are interfered at an angle, Eqs. 19/20 and/or Eq. 33 canbe applied where the intensities I₁ through I₄ represent the intensitieson neighboring pixels or bins of pixels with a 90° phase offset. In thecase that the sample and reference beams are interfered with parallelbeams, the equations can be applied where the intensities I₁ through I₄represent the intensities of the same pixel or bins of pixels, butmeasured at successive different optical phase delays, such as bychanging the phase delay of variable phase retarder 508. In addition itis possible to vary the phase relationship by moving one or more of themirrors in the reference path to change the reference path length. It isalso possible to rotate one or more mirror, for example 506 and/or 510to change the angle of interference. Variable attenuators (not shown)may be included in the sample and/or reference beam to substantiallymatch the sample and reference beam intensities.

FIG. 6 shows an alternate embodiment of a wide field setup for OPTIRoptical phase measurements. FIG. 6 is based on FIGS. 1 and 5 and whereidentical numerical callouts are used the associated descriptions fromFIGS. 1 /5 apply as appropriate. FIG. 6 illustrates a multi-cameraarrangement for wide-field quadrature phase detection. The beam paths ofFIG. 6 proceed the same as FIG. 5 until the reference beam emerges fromfocusing optic 508, at the second lens in the beam expander in thereference arm. A quarter wave plate 600 is inserted into the referencearm to create a circularly polarized reference beam 602. This beamrecombines with the sample beam 514 in beam splitter 604 typically anon-polarizing beam splitter. The recombined sample and reference beamsare divided by the beam splitter on two different paths 606 and 608. Ineach of these paths are polarizing beam splitters 610 and 612 thatdivide the combined reference and sample beams onto four paths to up tofour cameras 614, 616, 618, 620. Interference patterns appear at each ofthese four cameras surfaces. The arrangement of the quarter wave plate600 and the polarizing beam splitter ensures that each camera capturesthe interferograms at different phases, substantially 90° apart. Cameraframes can be captured synchronously with synchronized frame grabber 621ensuring high temporal correlation between the different cameras.Capturing synchronous frames substantially eliminates environmentalconcerns about vibration and/or temperature drift between the sample andreference paths. Since all frames are capture simultaneously any overallphase shifts can readily be determined using the signals from themultiple cameras. The quantitative phase and/or the differential phasedue to IR absorption can be determined using the methods describedabout, but in this case the I₁, I₂, I₃, and I₄ refer to the intensitiesof matching pixels on the four cameras, i.e. I₁ corresponds to a pixelintensity on camera 1, I₂ the corresponding pixel intensity on camera 2,etc., where each of the cameras are 90° apart in optical phase. Suchmulti-camera quadrature approaches are described for use withdifferential interference contrast microscopy as described for examplein (1) William C. Warger II, Judith A. Newmark, Bing Zhao, Carol M.Warner, and Charles A. DiMarzio “Accurate cell counts in live mouseembryos using optical quadrature and differential interference contrastmicroscopy”, Proc. SPIE 6090, Three-Dimensional and MultidimensionalMicroscopy: Image Acquisition and Processing XIII, 609009 (23 Feb.2006); https://doi.org/10.1117/12.644922; and (2) Willie S. Rockward,Anthony L. Thomas, Bing Zhao, and Charles A. DiMarzio, “Quantitativephase measurements using optical quadrature microscopy,” Appl. Opt. 47,1684-1696 (2008), both incorporated by reference.

FIG. 7 illustrates an alternative embodiment of optics for wide fieldoptical phase based OPTIR. FIG. 7 is based on FIG. 2A and whereidentical numerical callouts are used, the discussion associated withFIG. 2A applies as appropriate. FIG. 7 illustrates an alternative meansof dividing the sample and reference beam onto two paths. FIG. 7 is thesame as FIG. 2A in that the sample 202 is illuminated by an IR beam 204which excites molecular resonances in the sample which are read out byprobe beam 200. Probe beam passes through sample 202 and transmitted andscattered light is collected by focusing optic 206. As before this beamis magnified by focusing optic 212, typically a microscope tube lens.The beam 214 emerging from the tube lens or other focusing optic is thenincident on beam splitter 700. Unlike FIGS. 1-2 , the beam splitter 700in this case can be a non-polarizing beam splitter, for example simply apartially reflecting mirror that is inclined at a slight angle. Oneportion 702 of beam 214 is transmitted through beam splitter 700 whileanother portion 704 is diverted at twice the angle of the beam splitter.Beam 704 is then reflected by reflector 706, typically a “D-mirror” orpickoff mirror used to separate closely spaced beams. The net result isan angled beam 220 that is arranged at a deviated angle with respect tobeam 702. These two beams propagate just as beams 218 and 220 in FIG. 2Auntil they are recombined at the surface 236 of array detector/camera238. One other difference between the embodiment of FIG. 7 and FIG. 2Ais that the embodiment of FIG. 7 does not require the polarizationrotating element 244 of FIG. 2 since the two beams 702 and 704 are notseparated by polarization and thus maintain the same polarization. Theinterferogram at surface 236 can then be analyzed to extract changes inphase due to IR absorption of the sample as described in the variousalgorithms above.

FIG. 8 shows an alternative embodiment employing a modified form ofphase contrast microscopy with dynamic phase adjustment to perform widefield measurements of infrared absorption by a sample. Infrared beam 800is arranged to illuminate a region 802 of a sample 804, as describedpreviously associated with FIG. 1 and other previous figures. A phasecontrast microscope is arranged to illuminate a region of sample 804 atleast partially overlapping with the IR illuminated region 802.Specifically, an illuminating probe beam 806 is passed through anannulus 808 that produces a ring of illuminating probe light 810. Thislight ring 810 is then focused by focusing optic 812, typically amicroscope condenser to create a focused spot of probe light on thesample 804, at least partially overlapping with IR illuminated region802. Probe light striking sample 804 then can take one of two paths.Probe light that is undeflected by the sample follows path 814,expanding again into a ring of light that mirrors the illumination lightpattern. This is typically called the “direct” or “surround” light. Inaddition to the direct/surround light, a portion of the illuminatinglight is scattered by the sample through a wide array of angles. Aportion of this scattered light is collected by focusing optic 818,typically a microscope objective. The cone of scattered light collectedby optic 818 is indicated the dashed line labeled 816. Conventionalphase contrast microscopy arranges to interfere the direct/surroundlight with the scattered light as described below. The transmitteddirect light 814 and the scattered light 816 are collimated or otherwiserefocused by optic 818 (e.g., the microscope objective) and then passedthrough a phase ring 824. Phase ring 824 is generally divided intoregions with two different phase retardations. For example, regions 824a and 824 c can have one retardation value and 824 b can have a secondretardation value. The difference in phase retardation is generallyarranged to induce a 90° phase change between the direct/surround light820 and the scattered light 822 that passes through the phase ring 824.Both the direct/surround light are then focused by focusing optic 826(typically a microscope tube lens) to form an image 830 of the sample.At this image plane, the phase shifted direct/surround light interfereswith the scattered light to produce brightness contrast depending on thephase shifts induced in the scattered light by the sample. Consider forexample scattered light passing through a biological cell and incurringaround 90° of phase retardation due to the difference in index of thecell vs the surrounding media as described previously. When thedirect/surround light passes through thinner regions of the phase ring,its phase is advanced by roughly 90°, resulting in a total phase shiftof ˜180° between the direct/surround light and the scattered light. The180° phase shift results in destructive interference, thus creating adark image of the tops of the cells against a brighter background.Thinner regions of the cells will incur less of a phase change on thescattered beam, thus resulting in less destructive interference andcausing these regions to be brighter. Note that the brightness of theimage 830 does not have a simple relationship to the sample thickness.Very thin regions of a cell will be bright, thicker regions dark, butthen even thicker regions can become bright again when the optical pathdifference exceed 90°. This leads to contrast inversions that is onesource of phase contrast microscopy artifacts described in thebackground section. These contrast inversions, nonlinear sensitivity tothickness and other artifacts would cause significant problemsinterpreting infrared images and spectra if a camera sensor were placedat image plane 830. Specifically, the sensitivity of the IR absorptionmeasurement would depend in a complicated way on the sample thickness.And for some thicknesses the sensitivity of the IR absorptionmeasurement could in fact be zero. The rest of the optical path in FIG.8 and the following description provides a mean to overcome this issueand provide uniform sensitivity independent of the optical pathdifference.

To understand the issue and its resolution in more detail, consider thebrightness at a point in a phase contrast image. For the moment,consider just simple interference between the direct and scatteredlight. (Departures from this simple model will be discussed later.) Thegeneral form for the intensity of interfering waveforms is given by:

I=I _(d) +I _(s)+√{square root over (I _(d) I _(s))} cos ϕ  Eq. 35:

Where in this case I_(d) refers to the intensity of the direct light andI_(s) is the intensity of the scattered light, and ϕ is the relativephase between these two waves. Now the phase ring 824 introduces a 90°phase difference between the two waves, so Eq. 35 can be rewritten as:

I=I _(d) +I _(s)+√{square root over (I _(d) I _(s))} cos(ϕ_(s)+90°)=I_(d) +I _(s)−√{square root over (I _(d) I _(s))} sin ϕ_(s)  Eq. 36:

Where ϕ_(s) is the phase difference induced by the sample. (Note thatthat in some forms of phase contrast microscopy, the phase on the directlight is retarded instead of advanced, resulting in a change of sign inthe interference term.) In the case of photothermal excitation byabsorption of IR light, the sample phase ϕ_(s) will have a constant DCterm ϕ₀, dependent on the index of refraction and thickness of the givenregion of the sample and a small change δ that results from IRabsorption by the sample, i.e.:

ϕ_(s)=ϕ₀+δ  Eq. 37:

Inserting this into Eq. 34 for the “hot frame” (e.g. IR beam on) gives:

I _(h) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}sin(ϕ₀+δ)  Eq. 38:

Using the compound angle formula sin(A+B)=sin A cos B+cos A sin B, thisresults in:

I _(h) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}(sin ϕ₀ cosδ−cos ϕ₀ sin δ)  Eq. 39:

Using small angle approximations described earlier for the small phasechange δ gives:

I _(h) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}(sin ϕ₀−δ cosϕ₀)  Eq. 40:

And with no IR illumination, the “cold frame” intensity would be

I _(c) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))} sin ϕ₀  Eq.41:

Subtracting the cold frame intensity (Eq. 41) from the hot frameintensity (Eq. 40) gives:

I _(h) −I _(c)=√{square root over (I _(d) I _(s))}δ cos ϕ₀  Eq. 42:

This can be solved for the photothermal phase change δ:

$\begin{matrix}{\delta = \frac{I_{h} - I_{c}}{\sqrt{I_{d}I_{s}}\cos\phi_{0}}} & {{Eq}.43}\end{matrix}$

Eq. 43 illustrates the problem with simply placing a camera at imageplane 830. The issue is that the sensitivity to measuring thephotothermal phase change δ depends on the DC phase ϕ₀. The cos ϕ₀ termcan vary between ±1 where the sensitivity depends on the thickness andindex of refraction of the sample. Specifically, the DC phase change ϕ₀is given by:

$\begin{matrix}{\phi_{0} = {2{\pi( {n_{s} - n_{m}} )}\frac{t_{s}}{\lambda}}} & {{Eq}.44}\end{matrix}$

where n_(s) is the index of the sample, n_(m) is the index of thesurrounding media, t_(s) is the sample thickness, and λ is thewavelength of the illuminating probe beam. In the case of a biologicalcell accumulates a DC phase shift ϕ₀ around 90° as discussed earlier,the cos ϕ₀ term can be around zero, causing a singularity in thecalculation of the photothermal phase change. Thus, placing a camera atsample image plane 830 with no other modifications would result inhighly non-uniform sensitivity to the photothermal phase change δ.

To address this, a 4f relay system is included in FIG. 8 with a variablephase retarder to allow measurements at a plurality of phase values thatwill allow a continuous and consistent measurement of the photothermalphase change δ, for arbitrary values of the DC phase ϕ₀.

A first relay focusing optic 832 is placed nominally at a distancecorresponding to the focal length of optic 832, thus substantiallycollimating the direct/surround and scattered beams. The collimatedbeams then pass through a location addressable variable phase retarder836, for example a spatial light modulator. An annular retardationpattern is programed onto the variable phase retarder, substantiallymatching the aspect ratios of the annular rings in phase ring 824. (Notethat phase ring 824 can also be omitted and all phase adjustment can beprovided by variable phase retarder 836.) The pattern and/or phaseretardation amplitude is controlled by phase controller 838, for exampleby applying a pattern of different voltage levels to phase retardingelements of variable phase retarder 836. The direct and scattered beams842 and 840 emerging from the variable phase retarder now have a newtotal DC phase difference equal to ϕ₀+ϕ_(r) where ϕ_(r) is the phasechange introduced by the retarder. Both beams are then refocused bysecond relay focusing optic 844 (the 2nd lens in the 4f relay system)and then focused to form an interference image 848 on the surface ofcamera 850. Note that the 4f phase retardation system can also bearranged in reflection. For example, phase retarder 836 can be areflective spatial light modulator light a liquid crystal on silicon(LCOS) phase retarder. In this case the optical path of FIG. 8 would befolded, for example into a V-shaped configuration. Controller 852 may beused to synchronize phase adjustment steps with acquisition of imageframes from camera 850. Phase controller 838 and controller 852 may alsobe integrated into a single control unit in some embodiments. Inembodiments, any other actuatable fixed-pattern mask (such as an LCD ora physical obstruction) could be used, or any other structure thatselectively adds an optical path length of about ⅛ wavelength or more ofthe light used by the probe beam 806.

The camera 850 then records images at two or more optical phaseretardations, typically 90° apart. For example, if hot frames are takenwith 0, 90, 180 and 270 degree retardations, the resulting pixelintensities I_(h1), I_(h2), I_(h3), and I_(h4) are given by:

I _(h1) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}(sin ϕ₀−δ cosϕ₀)  Eq. 45:

I _(h2) =I _(d) +I _(s)−√{square root over (I _(d) I_(s))}(sin(ϕ₀+90°)−δ cos(ϕ₀+90°))  Eq. 46:

I _(h3) =I _(d) +I _(s)−√{square root over (I _(d) I_(s))}(sin(ϕ₀+180°)−δ cos(ϕ₀+180°))  Eq. 47:

I _(h4) =I _(d) +I _(s)−√{square root over (I _(d) I_(s))}(sin(ϕ₀+270°)−δ cos(ϕ₀+270°))  Eq. 48

Which in turn can be simplified to:

I _(h1) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}(sin ϕ₀−δ cosϕ₀)0°  Eq. 50:

I _(h2) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))}(cos ϕ₀+δ sinϕ₀)90°  Eq. 51:

I _(h3) =I _(d) +I _(s)+√{square root over (I _(d) I _(s))}(sin ϕ₀−δ cosϕ₀)180°  Eq. 52:

I _(h4) =I _(d) +I _(s)+√{square root over (I _(d) I _(s))}(cos ϕ₀−δ sinϕ₀)270°  Eq. 53:

Similarly, the pixel intensities of the cold frames (IR off) in 90°phase offsets can be written as:

I _(c1) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))} sin ϕ₀  Eq.54:

I _(c2) =I _(d) +I _(s)−√{square root over (I _(d) I _(s))} cos ϕ₀  Eq.55:

I _(c3) =I _(d) +I _(s)+√{square root over (I _(d) I _(s))} sin ϕ₀  Eq.56:

I _(c4) =I _(d) +I _(s)+√{square root over (I _(d) I _(s))} cos ϕ₀  Eq.57:

Subtracting Eq. 54 from Eq. 50 gives

I _(h1) −I _(c1)=√{square root over (I _(r) I _(s))}δ cos ϕ₀  Eq. 58:

Which can be solved to give:

$\begin{matrix}{\delta_{1} = \frac{I_{h1} - I_{c1}}{\sqrt{I_{d}I_{s}}\cos\phi_{0}}} & {{Eq}.59}\end{matrix}$

Where the subscript in the δ₁ term indicates that it was calculated withthe first phase retardation of 0°. Subtracting Eq. 55 from Eq. 51 gives

I _(h2) −I _(c2)−√{square root over (I _(d) I _(s))}δ₂ sin ϕ₀  Eq. 60:

Which can be solved to give:

$\begin{matrix}{\delta_{2} = {- \frac{I_{h2} - I_{c2}}{\sqrt{I_{d}I_{s}}\sin\phi_{0}}}} & {{Eq}.61}\end{matrix}$

Where the subscript in the δ₂ term indicates that it was calculated withthe second phase retardation of 90°. Equations 59 and 61 have differingdependencies on the DC phase ϕ₀, and each equation used independentlywould have singularities. It is possible to eliminate the phase term ϕ₀and hence eliminate the singularity. If measurements at the 0 and 90°phase offsets are done with a sufficiently short separation in time andunder the same conditions, the two photothermal difference amplitudesare the same, i.e. δ₁=δ₂=δ. (This condition can be met if themeasurements at 0/90° phase are performed within a time that is shortcompared to any significant drift in the measurement system.)Rearranging Eqs. 59 and 61 results in:

$\begin{matrix}{{\cos\phi_{0}} = \frac{I_{h1} - I_{c1}}{\delta\sqrt{I_{d}I_{s}}}} & {{Eq}.62}\end{matrix}$ $\begin{matrix}{{\sin\phi_{0}} = \frac{I_{c2} - I_{h2}}{\delta\sqrt{I_{d}I_{s}}}} & {{Eq}.63}\end{matrix}$

Using the identity, cos² ϕ₀+sin²ϕ=1 with Eqs. 62 and 63 gives:

$\begin{matrix}{{\frac{1}{I_{d}I_{s}}\lbrack {( \frac{I_{h1} - I_{c1}}{\delta} )^{2} + ( \frac{I_{c2} - I_{h2}}{\delta} )^{2}} \rbrack} = 1} & {{Eq}.64}\end{matrix}$

Which in turn can be solved to give:

$\begin{matrix}{\delta = {\frac{1}{\sqrt{I_{d}I_{s}}}\sqrt{\lbrack {( {I_{h1} - I_{c1}} )^{2} + ( {I_{c2} - I_{h2}} )^{2}} \rbrack}}} & {{Eq}.65}\end{matrix}$

The factor

$\frac{1}{\sqrt{I_{d}I_{s}}}$

is just a DC scaling factor and in some situations it is not necessaryto measure this. For example, if the measurement system intensities arerelatively stable and one wants to measure relative IR absorptionspectra vs position, it can be sufficient to simply measure the quantityhot minus cold frames at two phases 90° apart (e.g. (I_(h1)−I_(c1)) and(I_(h2)−I_(c2))). Note that the Eq. 65 has the form of a root meansquare (RMS) sum and is in fact the RMS sum of the in phase (0°) andquadrature (90°) photothermal difference images. This can then berepeated at a plurality of wavelengths of the IR source. Eq. 65 is asignificant result as it allows rapid calculation of a signal that isindicative of the IR absorption spectrum of the sample, without the needto measure the optical phase ϕ₀ or the I_(d) and I_(s) terms. In thecase it is desired to perform a more quantitative measurement of δ, itis possible to solve for the

$\frac{1}{\sqrt{I_{d}I_{s}}}$

term using combinations of Eqs. 54-57. There are three unknowns I_(d),I_(s) and ϕ₀, so using pixel values from at least three of Eqs 54-57 itis possible to solve for all the unknowns. One example is illustratedbelow in Eqs. 66-76.

Subtracting Eq. 54 from Eq. 56 and Eq. 55 from Eq. 57 gives:

I _(c3) −I _(c1)=2√{square root over (I _(d) I _(s))} sin ϕ₀  Eq. 66:

I _(c4) −I _(c2)=2√{square root over (I _(d) I _(s))} cos ϕ₀  Eq. 67:

Dividing Eq. 66 by Eq. 67 gives:

$\begin{matrix}{{\tan\phi_{0}} = \frac{I_{c1} - ( {I_{d} + I_{s}} )}{I_{c2} - ( {I_{d} + I_{s}} )}} & {{Eq}.68}\end{matrix}$

Which can be inverted to give:

$\begin{matrix}{\phi_{0} = {\tan^{- 1}( \frac{I_{c1} - ( {I_{d} + I_{s}} )}{I_{c2} - ( {I_{d} + I_{s}} )} )}} & {{Eq}.69}\end{matrix}$

Adding Eq. 54 and Eq 56 gives:

I _(c1) +I _(c3)=2(I _(d) +I _(s))  Eq. 70:

And:

$\begin{matrix}{( {I_{d} + I_{s}} ) = \frac{I_{c1} + I_{c3}}{2}} & {{Eq}.71}\end{matrix}$

Plugging Eq. 71 into Eq. 69 gives:

$\begin{matrix}{\phi_{0} = {\tan^{- 1}( \frac{{2I_{c1}} - I_{c1} - I_{c3}}{{2I_{c2}} - I_{c1} - I_{c3}} )}} & {{Eq}.72}\end{matrix}$

Note this intermediate result also provides a quantitative measurementof the DC phase if desired. Eq. 66 can be re-arranged to give:

$\begin{matrix}{\frac{1}{\sqrt{I_{d}I_{s}}} = \frac{2\sin\phi_{0}}{I_{c3} - I_{c1}}} & {{Eq}.73}\end{matrix}$

Plugging Eq. 72 into 73 gives:

$\begin{matrix}{\frac{1}{\sqrt{I_{d}I_{s}}} = \frac{2{\sin( {\tan^{- 1}( \frac{{2I_{c1}} - I_{c1} - I_{c3}}{{2I_{c2}} - I_{c1} - I_{c3}} )} )}}{I_{c3} - I_{c1}}} & {{Eq}.74}\end{matrix}$

Using the identity

${{\sin( {\tan^{- 1}u} )} = \frac{u}{\sqrt{1 + u^{2}}}},$

Eq. 71 can be rewritten as:

$\begin{matrix}{\frac{1}{\sqrt{I_{d}I_{s}}} = {\frac{2}{I_{c3} - I_{c1}}\frac{u}{\sqrt{1 + u^{2}}}}} & {{Eq}.75}\end{matrix}$

Where

$u = \frac{{2I_{c1}} - I_{c1} - I_{c3}}{{2I_{c2}} - I_{c1} - I_{c3}}$

Plugging this into Eq. 65 gives

$\begin{matrix}{\delta = {\frac{2}{I_{c3} - I_{c1}}\frac{u}{\sqrt{1 + u^{2}}}\sqrt{\lbrack {( {I_{h1} - I_{c1}} )^{2} + ( {I_{c2} - I_{h2}} )^{2}} \rbrack}}} & {{Eq}.76}\end{matrix}$

Note that it is also possible to extract the photothermal difference δby inverting Eq. 37 to give:

δ=ϕ_(s)−ϕ₀  Eq. 78:

This requires measuring the optical phase ϕ_(s) when the sample isilluminated by IR light and the phase ϕ₀ when the IR light is off (i.e.,hot and cold image frames). To extract the phase values, it is necessaryto measure the hot and cold images for two or more phase offsets of theinterferometer (e.g., 0° and 90°) and the phase values for example canbe extracted using the inverse tangent or atan 2 function. An advantage,of the scheme outlined in Eqs. 62-65, however, is that it requires nocomputation of the DC phase values ϕ₀ or ϕ_(s). The simple RMS sumcalculation of Eq. 65 can in general be computed much faster thaninverse tangents which can enable faster measurement times.

FIG. 9 illustrates an alternative embodiment using an epi-illuminationscheme such that the sample in imaged in a reflection modeconfiguration. This arrangement is desirable for samples that are opaqueto at least one of the IR and probe beams. FIG. 9 is based on FIG. 1 andwhere identical numerical callouts are used, the discussion associatedwith FIG. 1 applies as appropriate. Note that the epi-illuminationscheme described associated with FIG. 9 can also be applied to theembodiments of FIGS. 5-8 as well. As with the other figures, infraredsource 100 produces a beam of infrared radiation 102 that illuminates aregion 108 of sample 110, exciting molecular resonances in IR absorbingregions of the sample and causing local heating that will be mapped bythe probe beam. Probe beam source 101 emits a beam 103 of proberadiation that in this case is incident on a beam splitter 900. At leasta portion of the probe beam 103 is then incident on a focusing optic109. In this case focusing optic 109 is usually a microscope objectivewhich in this epi-configuration will be used for both illumination andcollection. In one embodiment, probe radiation 103 is focused onto theback focal plane of focusing optic 109 to create a wide area ofillumination on sample 110 at least partially overlapping the IRilluminated region 108. Light reflected and scattered from the sample isrecollected by focusing optic 109 (or alternately by another collectionoptic, not shown). If collected by focusing optic 109, the collectedlight returns to beam splitter 900 where at least a portion 111 of thereflected and scattered light is directed towards optional mirror 112where it passes into the common path interferometer setup as describedassociated with FIGS. 1 and 2 . To improve optical throughput beamsplitter 900 may be a polarizing beam splitter used in combination witha quarter waveplate (not shown) to separate the incoming and outgoingbeams with high efficiency. As before, pixel intensities collected atthe surface 138 of camera 132 due to interference of sample andreference beams are used to create a signal that is indicative of IRabsorption by the sample over a wide area. Thisepi-illumination/reflection mode scheme can also be applied to theMach-Zender approaches of FIGS. 5 and 6 , the beam splitter/D-mirrorapproach of FIG. 7 , and the phase contrast approach of FIG. 8 . In thecase of FIG. 7 , beam splitter 900 would be inserted between the phaseannulus 808 and the focusing optic 812.

FIG. 10 illustrates an alternative embodiment using a phase contrastdetection scheme. FIG. 10 is based on FIG. 8 and where identicalnumerical callouts are used, the discussion associated with FIG. 8applies as appropriate. As with FIG. 8 , the embodiment of FIG. 10starts with a ring of light 810 from an annulus 808, for example from aphase contrast condenser. As with FIG. 8 , light that strikes the sample804 can take one of two paths, path 814 for the “direct” or “surround”light and path 816 for scattered light. Light on both paths is collectedby objective 818. In a traditional phase contrast microscope, a phasering is placed at the back focal plane of objective 818. In thisembodiment, a 4f optical relay system (e.g. with focusing optics 826 and832) is used to relay an image of the back focal plane of the objective818 to a new location where a variable phase interferometer is placed.Specifically, light beams 1000 that exit the 4f relay system strike beamsplitter 1001 and split onto two different paths 1002 and 1003. A platebeam splitter is shown for beam splitter 1001, but a cube beam splittere.g. a polarizing beam splitter cube may be used instead. The light onpaths towards optical masks 1004 and 1005. Optical masks 1004 and 1005have complementary reflective patterns, shown in cross-section at theirapproximate location in 1004 a and 1005 a and separately in face onviews in 1004 b and 1005 b. The apparent thickness of the reflectivepatterns shown in cross-section is highly exaggerated for clarity inFIG. 10 . The reflective coating need only be thick enough to reflect asubstantial portion of the incident light beams 1002 and 1003. Thereflective pattern 1005 a on mask 1005 has a form similar to thattraditionally used at the back focal plane of a phase contrast objective(i.e. a mask that interacts primarily with the “direct” or unscatteredlight). Mask 1004 with pattern 1004 b is a substantially complementarymask that interacts primarily with the scattered light. As drawn theblack circular regions of mask patterns 1004 a and 1005 a representareas that are highly reflective and the white regions are either highlytransmissive or absorptive.

A key difference here is that in a traditional phase contrastmicroscope, the phase mask at the back focal plane of the objectiveintroduces a fixed optical phase shift (typically around 90°). Thearrangement of FIG. 10 , however, enables the creation and rapidadjustment of an arbitrary optical phase shift between the direct andscattered light. This is accomplished when the light on paths 1002 and1003 are reflected back through beam splitter 1001 and are recombinedonto optical path 1007 and then focused with focusing optic 844 onto thesurface of camera 850. An interferogram 848 thus appears on the surfaceof camera 850 where the interferogram comprises the optical interferencepattern of the light reflected back on path 1002 and the light reflectedbath on path 1003, with any optical phase offset between the two pathsintroduced by a difference in optical path length on paths 1002 and1003. A phase adjuster is used vary the phase of the opticalinterference pattern at the camera surface. For example, an actuator1006 may be used to adjust the relative position of optical mask 1005and/or optical mask 1004. The actuator can for example be apiezoelectric actuator, a voice coil actuator or any other actuatorcapable of providing precise relative motion of mask 1005 vs. mask 1004.Optional phase controller 838 can be used to generate control signals toadjust actuator 1006 to desired path length differences and hencedesired optical phase shifts. Phase controller 838 may generate one ormore voltages, currents, or other control signals to generate thedesired phase shift. Controller 852 may be used to synchronize phaseadjustment steps with acquisition of image frames from camera 850. Phasecontroller 838 and controller 852 may also be integrated into a singlecontrol unit in some embodiments. Image frames at camera 850 can then beacquired at a plurality of optical phases under the conditions of IRlight on and off, following the description associated with FIG. 8 .Notably, the optical phase can be adjusted extremely quickly in thiscase. For example, piezoelectric transducers are available withactuation frequencies in the kHz to hundreds of kHz or even MHz range,especially when only a small actuation range is required. To achieve a90° phase shift, it is only necessary to move one of the reflectivemasks 1004 or 1005 by λ/8, where λ is the wavelength of light used inthe phase contrast detection. For example, at 532 nm wavelength, only66.5 nm of motion is required for 90° phase shift. This is easilyachievable, for example with Thorlabs model PA2AB piezo actuator, whichhas a range of 700 nm and a resonant frequency of 1.35 MHz. Many othersuitable piezo actuators are available. Note that it may be desirable toinclude a variety of additional optical elements not shown to optimizethe performance of the interferometer. For example, in the case that apolarizing beamsplitter is used, half wave plates and quarter waveplates can be used to optimize the transmission of light from thedifferent interferometer arms to the camera. It can also be desirable toplace an attenuator in the path of the direct light 1003 to better matchthe light received by the camera on the direct and scattered lightpaths. (The scattered light is typically substantially less than thedirect light.) In the case of a plate beam splitter, it may be desirableto include a compensation plate in one of the interferometer arms tocompensate for the fact that the light goes through the thickness of thebeam splitter more times on one arm of the interferometer than theother. It is possible implement a single optical element that acts asboth compensator and attenuator.

The high actuation speed of the embodiment of FIG. 10 has a specificadvantage that it can enable adjustment extremely rapid adjustment ofthe optical phase of the interferometer of this phase contrast detectionsuch that the measurements at two or more optical phases can occurnearly simultaneously, or more specifically separated by short enoughtimes that there is minimal drift or vibration between the two arms ofthe interferometer. FIG. 11 shows an example timing diagram that canachieve photothermal measurements at two optical phases in rapidsuccession. Trace 1100 represents trigger pulses to initiate theacquisition of a camera frame. Trace 1102 shows a gating pulse to turnon and off IR light to illuminate the sample. (In practice the IR ongate may include many sub-pulses as infrared lasers can typically pulseat rates much faster than typical camera frame rates. For example, ahigh-speed camera may operate at 2000 frames per second, whereas aquantum cascade laser may by pulsed at 100 kHz or even MHz frequencies.As such, the IR source may provide many pulses per camera hot frame.)Trace 1104 represents the control signal alternating the optical phaseof the interferometer of FIG. 10 between two successive relative phasesϕ₁, and ϕ₂, such as 0° and 90°, though in alternative embodimentsdifferent relative phases could be used. The time difference between thetwo measurements at two optical phases can be very short. For example,consider acquiring images at camera 850 at a rate of 2000 frames persecond where the IR light is gated on and off every other frame as shownin trace 1102. In this case, the optical phase would be adjusted aftereach hot/cold image pair, such as at 1000 Hz. Thus, the measurementsbetween successive optical phase steps would be 1 msec, during whichtime the interferometer drift/vibration will be minimal. The use of apiezoelectric actuator or other high-speed actuator makes it possible toachieve the desired phase adjustments on such short time scales, forexample in less than 100 msec, less than 10 msec, or even less than 1msec. This approach is advantageous because it can provide phaseadjustments on time scales even faster than a typical pixelated spatiallight modulator and also at substantially reduced cost. It is alsopossible to invert the phase offsets and the IR on/off gating, forexample alternating the phase ever other camera image and then gatingthe IR on/off after each dual phase measurement. This latter schemewould then achieve 500 μsec between the two different phases and provideeven more immunity to interferometer drift/vibration. (Which approach ispreferable depends on the relative stability of the probe lightsource/microscope versus the interferometer.)

Optical Efficiency

The following section addresses optical efficiency which is a criticalfactor to enable high SNR measurements at high frame rates. Theembodiments described herein in FIGS. 1, 2, 5-9 employ opticallyefficient designs that arrange for optical throughputs ranging from 42%to 88%. A key to this efficiency is the use of non-diffractive beamsplitters to separate the sample and reference beams. For example, theembodiments of FIGS. 1-2 employ polarizing beam splitter prisms (e.g.Rochon or Wollaston gratings). The embodiments of FIGS. 1-2 can achievean optical throughput of around 44% on each of the sample and referencearm (88% total), accounting for reflection losses at optical componentsurface and throughput through the spatial filter. (Note this estimatedoes not account for the efficiency of collection or transmission of thescattered light because this is highly sample dependent.) The opticalthroughput of the configuration of FIG. 5 can achieve a total opticalthroughput around 42%. The primary reason for the lower opticalthroughput is the use of beam splitter 516 which discards roughly halfof the light that is reflected/transmitted in the downwards directionand thus not striking camera 132. A second camera, however, can beplaced below beam splitter 516 thus capturing light on the alternatepath, bringing the total optical efficiency to 84%.

Optical throughputs for the various configurations described herein aresummarized in the table below. This is significantly better than can beachieved by the diffractive beam separation approach described in MiuTamamitsu, Keiichiro Toda, Ryoichi Horisaki, and Takuro Ideguchi,“Quantitative phase imaging with molecular vibrational sensitivity,”Opt. Lett. 44, 3729-3732 (2019), https://doi.org/10.1364/OL.44.003729.The use of a Ronchi ruling to diffract the light for the sample andreference beam does not lead to high optical throughput, as can be seenin an analysis of transmission efficiency of Ronchi gratings asdescribed in James E. Harvey, Richard N. Pfisterer, “Understandingdiffraction grating behavior: including conical diffraction and Rayleighanomalies from transmission gratings,” Opt. Eng. 58(8) 087105 (28 Aug.2019) https://doi.org/10.1117/1.OE.58.8.087105. See for example FIG. 15and Table 2 of the Harvey reference. The use of diffractive beamseparation has a total optical efficiency of <35% because only around25% of the light is transmitted in the 0^(th) diffraction order of thereference path and around 10% into the +1 order. (50% of the light isentirely blocked by the grating, another 10% of the light is discardedin the −1 order. In practice other optical losses would provide a bestcase optical throughput of around 33%. This approach is especiallydisadvantageous from an optical efficiency standpoint considering thatonly 10% of the incident light that interacts with the sample makes itto the detector, whereas the embodiments described herein withnon-diffractive beam splitters have as much as >80% of the lightinteracting with the sample incident on the detector.

Embodiment Total optical throughput FIGS. 1-2 88% FIG. 5 42% (singlecamera) 84% (dual camera) FIG. 6 81% FIG. 7 88% FIG. 8 82% FIG. 9 80%Diffractive beam separation 33%

The embodiments described herein are exemplary. Modifications,rearrangements, substitute processes, alternative elements, etc. may bemade to these embodiments and still be encompassed within the teachingsset forth herein. One or more of the steps, processes, or methodsdescribed herein may be carried out by one or more processing and/ordigital devices, suitably programmed.

Depending on the embodiment, certain acts, events, or functions of anyof the method steps described herein can be performed in a differentsequence, can be added, merged, or left out altogether (e.g., not alldescribed acts or events are necessary for the practice of thealgorithm). Moreover, in certain embodiments, acts or events can beperformed concurrently, rather than sequentially.

The various illustrative logical blocks, optical and control elements,and method steps described in connection with the embodiments disclosedherein can be implemented as electronic hardware, computer software, orcombinations of both. To clearly illustrate this interchangeability ofhardware and software, various illustrative components, blocks, modules,and steps have been described above generally in terms of theirfunctionality. Whether such functionality is implemented as hardware orsoftware depends upon the particular application and design constraintsimposed on the overall system. The described functionality can beimplemented in varying ways for each particular application, but suchimplementation decisions should not be interpreted as causing adeparture from the scope of the disclosure.

The various illustrative logical blocks and modules described inconnection with the embodiments disclosed herein can be implemented orperformed by a machine, such as a processor configured with specificinstructions, a digital signal processor (DSP), an application specificintegrated circuit (ASIC), a field programmable gate array (FPGA) orother programmable logic device, discrete gate or transistor logic,discrete hardware components, or any combination thereof designed toperform the functions described herein. A processor can be amicroprocessor, but in the alternative, the processor can be acontroller, microcontroller, or state machine, combinations of the same,or the like. A processor can also be implemented as a combination ofcomputing devices, e.g., a combination of a DSP and a microprocessor, aplurality of microprocessors, one or more microprocessors in conjunctionwith a DSP core, or any other such configuration.

The elements of a method, process, or algorithm described in connectionwith the embodiments disclosed herein can be embodied directly inhardware, in a software module executed by a processor, or in acombination of the two. A software module can reside in RAM memory,flash memory, ROM memory, EPROM memory, EEPROM memory, registers, harddisk, a removable disk, a CD-ROM, or any other form of computer-readablestorage medium known in the art. An exemplary storage medium can becoupled to the processor such that the processor can read informationfrom, and write information to, the storage medium. In the alternative,the storage medium can be integral to the processor. The processor andthe storage medium can reside in an ASIC. A software module can comprisecomputer-executable instructions which cause a hardware processor toexecute the computer-executable instructions.

Conditional language used herein, such as, among others, “can,” “might,”“may,” “e.g.,” and the like, unless specifically stated otherwise, orotherwise understood within the context as used, is generally intendedto convey that certain embodiments include, while other embodiments donot include, certain features, elements, and/or states. Thus, suchconditional language is not generally intended to imply that features,elements and/or states are in any way required for one or moreembodiments or that one or more embodiments necessarily include logicfor deciding, with or without author input or prompting, whether thesefeatures, elements and/or states are included or are to be performed inany particular embodiment. The terms “comprising,” “including,”“having,” “involving,” and the like are synonymous and are usedinclusively, in an open-ended fashion, and do not exclude additionalelements, features, acts, operations, and so forth. Also, the term “or”is used in its inclusive sense (and not in its exclusive sense) so thatwhen used, for example, to connect a list of elements, the term “or”means one, some, or all of the elements in the list.

Disjunctive language such as the phrase “at least one of X, Y or Z,”unless specifically stated otherwise, is otherwise understood with thecontext as used in general to present that an item, term, etc., may beeither X, Y or Z, or any combination thereof (e.g., X, Y and/or Z).Thus, such disjunctive language is not generally intended to, and shouldnot, imply that certain embodiments require at least one of X, at leastone of Y or at least one of Z to each be present.

Unless otherwise explicitly stated, articles such as “a” or “an” shouldgenerally be interpreted to include one or more described items.Accordingly, phrases such as “a device configured to” are intended toinclude one or more recited devices. Such one or more recited devicescan also be collectively configured to carry out the stated recitations.For example, “a processor configured to carry out recitations A, B andC” can include a first processor configured to carry out recitation Aworking in conjunction with a second processor configured to carry outrecitations B and C.

Any incorporation by reference of documents above is limited such thatno subject matter is incorporated that is contrary to the explicitdisclosure herein. Any incorporation by reference of documents above isfurther limited such that no claims included in the documents areincorporated by reference herein. Any incorporation by reference ofdocuments above is yet further limited such that any definitionsprovided in the documents are not incorporated by reference hereinunless expressly included herein.

For purposes of interpreting the claims, it is expressly intended thatthe provisions of Section 112, sixth paragraph of 35 U.S.C. are not tobe invoked unless the specific terms “means for” or “step for” arerecited in a claim.

While the above detailed description has shown, described, and pointedout novel features as applied to illustrative embodiments, it will beunderstood that various omissions, substitutions, and changes in theform and details of the devices or methods illustrated can be madewithout departing from the spirit of the disclosure. As will berecognized, certain embodiments described herein can be embodied withina form that does not provide all of the features and benefits set forthherein, as some features can be used or practiced separately fromothers. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

1. A system for infrared analysis over a wide field area of a sample,the system comprising: an infrared source configured to illuminate aregion of the sample with a pump beam of infrared radiation to create ininfrared illuminated region; a probe radiation source configured togenerate a probe beam that illuminates a wide field region of the samplewherein the wide field region is at least 50 microns in diameter and atleast partially overlaps the infrared illuminated region of the sample;a collection optic arranged to collect at least a portion of the probebeam that has interacted with the sample; a first optical systemcomprising a non-diffractive beam splitter that divides the probe beamcollected from the sample onto at least two paths, a first path for areference beam and a second path for a sample beam; a second opticalsystem comprising a 4f optical relay system and arranged to spatiallyfilter the reference beam and create an inteferogram formed between thereference beam and the sample beam as part of an image of the region ofthe sample on a surface of an array detector that is captured as animage frame of the wide field region of the sample; and an analyzerconfigured to analyze the image frame to determine signals indicative ofphotothermal infrared absorption over the wide field area of the sample.2. The system of claim 1 wherein the array detector is a camera having aframe rate for capturing successive image frames of the wide field areaof the sample of at least 100 frames per second.
 3. The system of claim1 wherein the first optic system comprises: an illumination portion thatincludes light from a collected probe beam that was transmitted throughthe sample in a substantially undeflected state; and an imaging portionthat includes light from the collected probe beam that is at least oneof scattered, refracted, and reflected at the sample; wherein theillumination portion and the imaging portion each include image datacorresponding to characteristics of the sample, and wherein thenon-diffractive beam splitter divides the probe beam such that theillumination portion comprises the reference beam and the imagingportion comprises the sample beam.
 4. The system of claim 1 wherein thesecond optic system comprises a system of lenses configured to: focus afirst portion of the illumination portion having a first polarization ata spatial filter such that the image data corresponding to thecharacteristics of the sample in a first portion of the illuminationportion is removed; focus a second portion of the illumination portionhaving a second polarization different from the first polarization at apolarization rotator such that a second portion of the illuminationportion retains the image data corresponding to characteristics of thesample; direct a first portion of the imaging portion having the firstpolarization at the spatial filter such that a majority of the firstportion of the imaging portion is blocked; direct a second portion ofthe imaging portion having the second polarization at the polarizationrotator such that the second portion of the illumination portion retainsthe image data corresponding to the characteristics of the sample; andinterfere the first portion of the illumination portion, the secondportion of the illumination portion, the first portion of the imagingportion that is not blocked, and the second portion of the imagingportion to form a recombined beam as the image of the region of thesample on the surface of the array detector that is captured as theimage frame of the wide field area of the sample, wherein thepolarization rotator is configured to impart the first polarization onthe second portion of the illumination portion and the second portion ofthe imaging portion at the recombined beam.
 5. A system for infraredanalysis over a wide field area of a sample, the system comprising: aninfrared source configured to illuminate a region of the sample with apump beam of infrared radiation to create in infrared illuminatedregion; a probe radiation source configured to generate a probe beamthat illuminates a wide field region of the sample wherein the widefield region is at least 50 microns in diameter and at least partiallyoverlaps the infrared illuminated region of the sample; a collectionoptic arranged to collect at least a portion of the probe beam that hasinteracted with the sample; a first optical system comprising anon-diffractive beam splitter that divides the probe beam collected fromthe sample onto at least two paths, a first path for a reference beamand a second path for a sample beam; a second optical system comprisinga 4f optical relay system and arranged to spatially filter the referencebeam and create an interferogram formed between the reference beam andthe sample beam as part of an image of the region of the sample on asurface of an array detector that is captured as an image frame of thewide field region of the sample; and an analyzer configured to analyzethe image frame to determine signals indicative of photothermal infraredabsorption over the wide field area of the sample, wherein the arraydetector is a camera and the first optic system and the second opticsystem are configured to provide an optical throughput efficiency of atleast 50%.
 6. The system of claim 5 wherein the non-diffractive beamsplitter comprises at least one of a Wollaston prism, a Rochon prism, areflective beam splitter or a polarizing beam splitter.
 7. (canceled) 8.The system of claim 5 wherein the first optic system comprises: anillumination portion that includes light from the probe beam that wastransmitted through the sample in a substantially undeflected state; andan imaging portion that includes light from the probe beam that is atleast one of scattered, refracted, and reflected at the sample; whereinthe illumination portion and the imaging portion each include image datacorresponding to characteristics of the sample, and wherein thenon-diffractive beam splitter divides the probe beam such that theillumination portion comprises the reference beam and the imagingportion comprises the sample beam.
 9. The system of claim 5 wherein thesecond optic system comprises a system of lenses configured to: focus afirst portion of the illumination portion having a first polarization ata spatial filter such that the image data corresponding to thecharacteristics of the sample in a first portion of the illuminationportion is removed; focus a second portion of the illumination portionhaving a second polarization different from the first polarization at apolarization rotator such that a second portion of the illuminationportion retains the image data corresponding to characteristics of thesample; direct a first portion of the imaging portion having the firstpolarization at the spatial filter such that a majority of the firstportion of the imaging portion is blocked; direct a second portion ofthe imaging portion having the second polarization at the polarizationrotator such that the second portion of the illumination portion retainsthe image data corresponding to the characteristics of the sample; andinterfere the first portion of the illumination portion, the secondportion of the illumination portion, the first portion of the imagingportion that is not blocked, and the second portion of the imagingportion to form a recombined beam as the image of the region of thesample on the surface of the array detector that is captured as theimage frame of the wide field area of the sample, wherein thepolarization rotator is configured to impart the first polarization onthe second portion of the illumination portion and the second portion ofthe imaging portion at the recombined beam.
 10. A system for infraredanalysis over a wide field area of a sample, the system comprising: aninfrared source configured to illuminate a region of the sample with apump beam of infrared radiation to create an infrared illuminatedregion; a probe radiation source configured to generate an annular probebeam that illuminates a wide field region of the sample wherein the widefield region is at least 50 microns in diameter and at least partiallyoverlaps the infrared illuminated region of the sample; a collectionoptic arranged to collect the probe beam from the sample; an opticalsystem comprising a 4f optical relay system including at least onevariable phase retarder configured with an annular phase shift patternto create phase contrast interference between direct/surroundillumination probe from the light beam that passes through the samplewith light from the probe beam scattered by the sample to create aninterference image on a surface of an array detector that is captured asan image frame of the wide field region of the sample; and an analyzerconfigured to analyze the image frame to determine signals indicative ofphotothermal infrared absorption over the wide field area of the sample.11. The system of claim 10 further comprising a camera having a framerate for capturing successive image frames of the wide field area of thesample of at least 100 frames per second, wherein the camera isconfigured to receive the image frame of the wide field region of thesample.
 12. The system of claim 10 wherein the annular probe beam ispulsed at a rate at least equal to the frame rate of the camera.
 13. Thesystem of claim 10 wherein the optical system further comprises anon-diffractive beam splitter configured to divides the probe beam suchthat an illumination portion comprises the reference beam and an imagingportion comprises the sample beam.
 14. A system for infrared analysisover a wide field area of a sample, the system comprising: an infraredsource configured to illuminate a region of the sample with a pump beamof infrared radiation to create in infrared illuminated region; a proberadiation source configured to generate a probe beam that illuminates aregion of the sample that at least partially overlaps the infraredilluminated region of the sample; a collection optic arranged to collectat least a portion of probe beam radiation after interacting with thesample; a beam splitter that divides the collected probe beam onto atleast two paths, including a first path and a second path; a firstoptical mask on the first path having a first reflection patternarranged to substantially reflect direct light comprising collectedprobe beam radiation that has not been substantially deflected by thesample; a second optical mask on the second path having a secondreflection pattern that is a counterpart to the first reflection patternand is arranged to substantially reflect scattered light comprisingcollected probe radiation that has been scattered by the sample; acamera configured to capture image frames corresponding tointerferograms between the reference beam and the sample beam; a phaseadjuster arranged to adjust a relative optical phase between directlight and scattered light reflected from the first and secondreflectors; and an analyzer configured to analyze the image frames at atleast two relative optical phases to determine signals indicative ofphotothermal infrared absorption over the wide field area of the sample.15. The system of claim 14, wherein the phase adjuster comprises anactuator that moves at least one of first or second mask.
 16. The systemof claim 15, wherein the actuator comprises at least one of apiezoelectric and voice coil actuator.
 17. The system of claim 14,wherein the actuator is configured to adjust the relative optical phasesuch that each frame has a duration of less than 100 msec, morepreferably less than 10 msec, and even more preferably less than 1 msec.18. The system of claim 14, wherein the camera is configured to captureat least two image frames having a phase offset that is substantially 90degrees.
 19. The system of claim 14 wherein area of the sampleilluminated by the probe beam the is at least 50 microns in diameter.